Stress-related polynucleotides and polypeptides in plants

ABSTRACT

The invention relates to plant transcription factor polypeptides, polynucleotides that encode them, homologs from a variety of plant species, and methods of using the polynucleotides and polypeptides to produce transgenic plants having advantageous properties compared to a reference plant. Sequence information related to these polynucleotides and polypeptides can also be used in bioinformatic search methods and is also disclosed.

This application claims the benefit of U.S. Provisional Application No.60/310,847, filed Aug. 9, 2001, U.S. Provisional Application No.60/336,049, filed Nov. 19, 2001, and U.S. Provisional Application No.60/338,692, filed Dec. 11, 2001; and, this application is acontinuation-in-part of prior U.S. Non-provisional Application No.09/837,944, filed Apr. 18, 2001 (abandoned), and prior U.S.Non-provisional Application No. 10/171,468, filed Jun. 14, 2002(abandoned), the entire contents of which are hereby incorporated byreference.

The claimed invention, in the field of functional genomics and thecharacterization of plant genes for the improvement of plants, was madeby or on behalf of Mendel Biotechnology, Inc. and Monsanto Corporationas a result of activities undertaken within the scope of a jointresearch agreement, said agreement having been in effect on or beforethe date the claimed invention was made.

FIELD OF THE INVENTION

This invention relates to the field of plant biology. More particularly,the present invention pertains to compositions and methods forphenotypically modifying a plant.

INTRODUCTION

A plant's traits, such as its biochemical, developmental, or phenotypiccharacteristics, may be controlled through a number of cellularprocesses. One important way to manipulate that control is throughtranscription factors—proteins that influence the expression of aparticular gene or sets of genes. Transformed and transgenic plants thatcomprise cells having altered levels of at least one selectedtranscription factor, for example, possess advantageous or desirabletraits. Strategies for manipulating traits by altering a plant cell'stranscription factor content can therefore result in plants and cropswith commercially valuable properties. Applicants have identifiedpolynucleotides encoding transcription factors, developed numeroustransgenic plants using these polynucleotides, and have analyzed theplants for a variety of important traits. In so doing, applicants haveidentified important polynucleotide and polypeptide sequences forproducing commercially valuable plants and crops as well as the methodsfor making them and using them. Other aspects and embodiments of theinvention are described below and can be derived from the teachings ofthis disclosure as a whole.

BACKGROUND OF THE INVENTION

Transcription factors can modulate gene expression, either increasing ordecreasing (inducing or repressing) the rate of transcription. Thismodulation results in differential levels of gene expression at variousdevelopmental stages, in different tissues and cell types, and inresponse to different exogenous (e.g., environmental) and endogenousstimuli throughout the life cycle of the organism.

Because transcription factors are key controlling elements of biologicalpathways, altering the expression levels of one or more transcriptionfactors can change entire biological pathways in an organism. Forexample, manipulation of the levels of selected transcription factorsmay result in increased expression of economically useful proteins ormetabolic chemicals in plants or to improve other agriculturallyrelevant characteristics. Conversely, blocked or reduced expression of atranscription factor may reduce biosynthesis of unwanted compounds orremove an undesirable trait. Therefore, manipulating transcriptionfactor levels in a plant offers tremendous potential in agriculturalbiotechnology for modifying a plant's traits.

The present invention provides novel transcription factors useful formodifying a plant's phenotype in desirable ways.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a recombinant polynucleotidecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence encoding a polypeptide comprising apolypeptide sequence selected from those of the Sequence Listing, SEQ IDNOs: 2 to 2N, where N=2–123, or those listed in Table 4, or acomplementary nucleotide sequence thereof; (b) a nucleotide sequenceencoding a polypeptide comprising a variant of a polypeptide of (a)having one or more, or between 1 and about 5, or between 1 and about 10,or between 1 and about 30, conservative amino acid substitutions; (c) anucleotide sequence comprising a sequence selected from those of SEQ IDNOs: 1 to (2N−1), where N=2–123, or those included in Table 4, or acomplementary nucleotide sequence thereof; (d) a nucleotide sequencecomprising silent substitutions in a nucleotide sequence of (c); (e) anucleotide sequence which hybridizes under stringent conditions oversubstantially the entire length of a nucleotide sequence of one or moreof: (a), (b), (c), or (d); (f) a nucleotide sequence comprising at least10 or 15, or at least about 20, or at least about 30 consecutivenucleotides of a sequence of any of (a)–(e), or at least 10 or 15, or atleast about 20, or at least about 30 consecutive nucleotides outside ofa region encoding a conserved domain of any of (a)–(e); (g) a nucleotidesequence comprising a subsequence or fragment of any of (a)–(f), whichsubsequence or fragment encodes a polypeptide having a biologicalactivity that modifies a plant's characteristic, functions as atranscription factor, or alters the level of transcription of a gene ortransgene in a cell; (h) a nucleotide sequence having at least 31%sequence identity to a nucleotide sequence of any of (a)–(g); (i) anucleotide sequence having at least 60%, or at least 70%, or at least80%, or at least 90%, or at least 95% sequence identity to a nucleotidesequence of any of (a)–(g) or a 10 or 15 nucleotide, or at least about20, or at least about 30 nucleotide region of a sequence of (a)–(g) thatis outside of a region encoding a conserved domain; (j) a nucleotidesequence that encodes a polypeptide having at least 31% sequenceidentity to a polypeptide listed in Table 4, or the Sequence Listing;(k) a nucleotide sequence which encodes a polypeptide having at least60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%sequence identity to a polypeptide listed in Table 4, or the SequenceListing; and (l) a nucleotide sequence that encodes a conserved domainof a polypeptide having at least 85%, or at least 90%, or at least 95%,or at least 98% sequence identity to a conserved domain of a polypeptidelisted in Table 4, or the Sequence Listing. The recombinantpolynucleotide may further comprise a constitutive, inducible, ortissue-specific promoter operably linked to the nucleotide sequence. Theinvention also relates to compositions comprising at least two of theabove-described polynucleotides.

In a second aspect, the invention comprises an isolated or recombinantpolypeptide comprising a subsequence of at least about 10, or at leastabout 15, or at least about 20, or at least about 30 contiguous aminoacids encoded by the recombinant or isolated polynucleotide describedabove, or comprising a subsequence of at least about 8, or at leastabout 12, or at least about 15, or at least about 20, or at least about30 contiguous amino acids outside a conserved domain.

In a third aspect, the invention comprises an isolated or recombinantpolynucleotide that encodes a polypeptide that is a paralog of theisolated polypeptide described above. In one aspect, the invention is aparalog which, when expressed in Arabidopsis, modifies a trait of theArabidopsis plant.

In a fourth aspect, the invention comprises an isolated or recombinantpolynucleotide that encodes a polypeptide that is an ortholog of theisolated polypeptide described above. In one aspect, the invention is anortholog which, when expressed in Arabidopsis, modifies a trait of theArabidopsis plant.

In a fifth aspect, the invention comprises an isolated polypeptide thatis a paralog of the isolated polypeptide described above. In one aspect,the invention is a paralog which, when expressed in Arabidopsis,modifies a trait of the Arabidopsis plant.

In a sixth aspect, the invention comprises an isolated polypeptide thatis an ortholog of the isolated polypeptide described above. In oneaspect, the invention is an ortholog which, when expressed inArabidopsis, modifies a trait of the Arabidopsis plant.

The present invention also encompasses transcription factor variants. Apreferred transcription factor variant is one having at least 40% aminoacid sequence identity, a more preferred transcription factor variant isone having at least 50% amino acid sequence identity and a mostpreferred transcription factor variant is one having at least 65% aminoacid sequence identity to the transcription factor amino acid sequencesSEQ ID NOs: 2 to 2N, where N=2–123, and which contains at least onefunctional or structural characteristic of the transcription factoramino acid sequences. Sequences having lesser degrees of identity butcomparable biological activity are considered to be equivalents.

In another aspect, the invention is a transgenic plant comprising one ormore of the above-described isolated or recombinant polynucleotides. Inyet another aspect, the invention is a plant with altered expressionlevels of a polynucleotide described above or a plant with alteredexpression or activity levels of an above-described polypeptide.Further, the invention is a plant lacking a nucleotide sequence encodinga polypeptide described above or substantially lacking a polypeptidedescribed above. The plant may be any plant, including, but not limitedto, Arabidopsis, mustard, soybean, wheat, corn, potato, cotton, rice,oilseed rape, sunflower, alfalfa, sugarcane, turf, banana, blackberry,blueberry, strawberry, raspberry, cantaloupe, carrot, cauliflower,coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon,onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweetcorn, tobacco, tomato, watermelon, rosaceous fruits, vegetablebrassicas, and mint or other labiates. In yet another aspect, theinventions is an isolated plant material of a plant, including, but notlimited to, plant tissue, fruit, seed, plant cell, embryo, protoplast,pollen, and the like. In yet another aspect, the invention is atransgenic plant tissue culture of regenerable cells, including, but notlimited to, embryos, meristematic cells, microspores, protoplast,pollen, and the like.

In yet another aspect the invention is a transgenic plant comprising oneor more of the above described polynucleotides wherein the encodedpolypeptide is expressed and regulates transcription of a gene.

In a further aspect the invention provides a method of using thepolynucleotide composition to breed a progeny plant from a transgenicplant including crossing plants, producing seeds from transgenic plants,and methods of breeding using transgenic plants, the method comprisingtransforming a plant with the polynucleotide composition to create atransgenic plant, crossing the transgenic plant with another plant,selecting seed, and growing the progeny plant from the seed.

In a further aspect, the invention provides a progeny plant derived froma parental plant wherein said progeny plant exhibits at least three foldgreater messenger RNA levels than said parental plant, wherein themessenger RNA encodes a DNA-binding protein which is capable of bindingto a DNA regulatory sequence and inducing expression of a plant traitgene, wherein the progeny plant is characterized by a change in theplant trait compared to said parental plant. In yet a further aspect,the progeny plant exhibits at least ten fold greater messenger RNAlevels compared to said parental plant. In yet a further aspect, theprogeny plant exhibits at least fifty fold greater messenger RNA levelscompared to said parental plant.

In a further aspect, the invention relates to a cloning or expressionvector comprising the isolated or recombinant polynucleotide describedabove or cells comprising the cloning or expression vector.

In yet a further aspect, the invention relates to a composition producedby incubating a polynucleotide of the invention with a nuclease, arestriction enzyme, a polymerase; a polymerase and a primer; a cloningvector, or with a cell.

Furthermore, the invention relates to a method for producing a planthaving a modified trait. The method comprises altering the expression ofan isolated or recombinant polynucleotide of the invention or alteringthe expression or activity of a polypeptide of the invention in a plantto produce a modified plant, and selecting the modified plant for amodified trait. In one aspect, the plant is a monocot plant. In anotheraspect, the plant is a dicot plant. In another aspect the recombinantpolynucleotide is from a dicot plant and the plant is a monocot plant.In yet another aspect the recombinant polynucleotide is from a monocotplant and the plant is a dicot plant. In yet another aspect therecombinant polynucleotide is from a monocot plant and the plant is amonocot plant. In yet another aspect the recombinant polynucleotide isfrom a dicot plant and the plant is a dicot plant.

In another aspect, the invention is a transgenic plant comprising anisolated or recombinant polynucleotide encoding a polypeptide whereinthe polypeptide is selected from the group consisting of SEQ ID NOs:2—2N where N=2–123. In yet another aspect, the invention is a plant withaltered expression levels of a polypeptide described above or a plantwith altered expression or activity levels of an above-describedpolypeptide. Further, the invention is a plant lacking a polynucleotidesequence encoding a polypeptide described above or substantially lackinga polypeptide described above. The plant may be any plant, including,but not limited to, Arabidopsis, mustard, soybean, wheat, corn, potato,cotton, rice, oilseed rape, sunflower, alfalfa, sugarcane, turf, banana,blackberry, blueberry, strawberry, raspberry, cantaloupe, carrot,cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce,mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach,squash, sweet corn, tobacco, tomato, watermelon, rosaceous fruits,vegetable brassicas, and mint or other labiates. In yet another aspect,the inventions is an isolated plant material of a plant, including, butnot limited to, plant tissue, fruit, seed, plant cell, embryo,protoplast, pollen, and the like. In yet another aspect, the inventionis a transgenic plant tissue culture of regenerable cells, including,but not limited to, embryos, meristematic cells, microspores,protoplast, pollen, and the like.

In another aspect, the invention relates to a method of identifying afactor that is modulated by or interacts with a polypeptide encoded by apolynucleotide of the invention. The method comprises expressing apolypeptide encoded by the polynucleotide in a plant; and identifying atleast one factor that is modulated by or interacts with the polypeptide.In one embodiment the method for identifying modulating or interactingfactors is by detecting binding by the polypeptide to a promotersequence, or by detecting interactions between an additional protein andthe polypeptide in a yeast two hybrid system, or by detecting expressionof a factor by hybridization to a microarray, subtractive hybridization,or differential display.

In yet another aspect, the invention is a method of identifying amolecule that modulates activity or expression of a polynucleotide orpolypeptide of interest. The method comprises placing the molecule incontact with a plant comprising the polynucleotide or polypeptideencoded by the polynucleotide of the invention and monitoring one ormore of the expression level of the polynucleotide in the plant, theexpression level of the polypeptide in the plant, and modulation of anactivity of the polypeptide in the plant.

In yet another aspect, the invention relates to an integrated system,computer or computer readable medium comprising one or more characterstrings corresponding to a polynucleotide of the invention, or to apolypeptide encoded by the polynucleotide. The integrated system,computer or computer readable medium may comprise a link between one ormore sequence strings to a modified plant trait.

In yet another aspect, the invention is a method for identifying asequence similar or homologous to one or more polynucleotides of theinvention, or one or more polypeptides encoded by the polynucleotides.The method comprises providing a sequence database, and querying thesequence database with one or more target sequences corresponding to theone or more polynucleotides or to the one or more polypeptides toidentify one or more sequence members of the database that displaysequence similarity or homology to one or more of the one or more targetsequences.

The method may further comprise of linking the one or more of thepolynucleotides of the invention, or encoded polypeptides, to a modifiedplant phenotype.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING, TABLES, AND FIGURE

The Sequence Listing provides exemplary polynucleotide and polypeptidesequences of the invention. The traits associated with the use of thesequences are included in the Examples.

CD-ROM1 (Copy 1) is a read-only memory computer-readable compact discand contains a copy of the Sequence Listing in ASCII text format. TheSequence Listing is named “SeqList_(—)0036-1US txt”, file creation dateof Aug. 9, 2002, and is 896 kilobytes in size. The copies of theSequence Listing on the CD-ROM disc are hereby incorporated by referencein their entirety.

CD-ROM 2 (Copy 2) is an exact copy of CD-R1 (Copy 1).

CD-ROM3 contains a CRF copy of the Sequence Listing as a text (.txt)file. The CRF copy of the Sequence Listing is named“SeqList_(—)0036-1US.txt”, is 896 kilobytes in size and was created onAug. 9, 2002.

Table 4 shows the polynucleotides and polypeptides identified by SEQ IDNO; Mendel Gene ID No.; conserved domain of the polypeptide; and if thepolynucleotide was tested in a transgenic assay. The first column showsthe polynucleotide SEQ ID NO; the second column shows the Mendel Gene IDNo., GID; the third column shows the trait(s) resulting from the knockout or overexpression of the polynucleotide in the transgenic plant; thefourth column shows the category of the trait; the fifth column showsthe transcription factor family to which the polynucleotide belongs; thesixth column (“Comment”), includes specific effects and utilitiesconferred by the polynucleotide of the first column; the seventh columnshows the SEQ ID NO of the polypeptide encoded by the polynucleotide;and the eighth column shows the amino acid residue positions of theconserved domain in amino acid (AA) co-ordinates.

Table 5 lists a summary of orthologous and homologous sequencesidentified using BLAST (tblastx program). The first column shows thepolynucleotide sequence identifier (SEQ ID NO), the second column showsthe corresponding cDNA identifier (Gene ID), the third column shows theorthologous or homologous polynucleotide GenBank Accession Number (TestSequence ID), the fourth column shows the calculated probability valuethat the sequence identity is due to chance (Smallest Sum Probability),the fifth column shows the plant species from which the test sequencewas isolated (Test Sequence Species), and the sixth column shows theorthologous or homologous test sequence GenBank annotation (TestSequence GenBank Annotation).

FIG. 1 shows a phylogenic tree of related plant families adapted fromDaly et al. (2001 Plant Physiology 127:1328–1333).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

In an important aspect, the present invention relates to polynucleotidesand polypeptides, e.g. for modifying phenotypes of plants. Throughoutthis disclosure, various information sources are referred to and/or arespecifically incorporated. The information sources include scientificjournal articles, patent documents, textbooks, and World Wide Webbrowser-inactive page addresses, for example. While the reference tothese information sources clearly indicates that they can be used by oneof skill in the art, applicants specifically incorporate each and everyone of the information sources cited herein, in their entirety, whetheror not a specific mention of “incorporation by reference” is noted. Thecontents and teachings of each and every one of the information sourcescan be relied on and used to make and use embodiments of the invention.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “aplant” includes a plurality of such plants, and a reference to “astress” is a reference to one or more stresses and equivalents thereofknown to those skilled in the art, and so forth.

The polynucleotide sequences of the invention encode polypeptides thatare members of well-known transcription factor families, including planttranscription factor families, as disclosed in Table 4. Generally, thetranscription factors encoded by the present sequences are involved incell differentiation and proliferation and the regulation of growth.Accordingly, one skilled in the art would recognize that by expressingthe present sequences in a plant, one may change the expression ofautologous genes or induce the expression of introduced genes. Byaffecting the expression of similar autologous sequences in a plant thathave the biological activity of the present sequences, or by introducingthe present sequences into a plant, one may alter a plant's phenotype toone with improved traits. The sequences of the invention may also beused to transform a plant and introduce desirable traits not found inthe wild-type cultivar or strain. Plants may then be selected for thosethat produce the most desirable degree of over- or underexpression oftarget genes of interest and coincident trait improvement.

The sequences of the present invention may be from any species,particularly plant species, in a naturally occurring form or from anysource whether natural, synthetic, semi-synthetic or recombinant. Thesequences of the invention may also include fragments of the presentamino acid sequences. In this context, a “fragment” refers to a fragmentof a polypeptide sequence which is at least 5 to about 15 amino acids inlength, most preferably at least 14 amino acids, and which retain somebiological activity of a transcription factor. Where “amino acidsequence” is recited to refer to an amino acid sequence of a naturallyoccurring protein molecule, “amino acid sequence” and like terms are notmeant to limit the amino acid sequence to the complete native amino acidsequence associated with the recited protein molecule.

As one of ordinary skill in the art recognizes, transcription factorscan be identified by the presence of a region or domain of structuralsimilarity or identity to a specific consensus sequence or the presenceof a specific consensus DNA-binding site or DNA-binding site motif (see,for example, Riechmann et al., (2000) Science 290: 2105–2110). The planttranscription factors may belong to one of the following transcriptionfactor families: the AP2 (APETALA2) domain transcription factor family(Riechmann and Meyerowitz (1998) Biol. Chem. 379:633–646); the MYBtranscription factor family (Martin and Paz-Ares, (1997) Trends Genet.13:67–73); the MADS domain transcription factor family (Riechmann andMeyerowitz (1997) Biol. Chem. 378:1079–1101); the WRKY protein family(Ishiguro and Nakamura (1994) Mol. Gen. Genet. 244:563–571); theankyrin-repeat protein family (Zhang et al. (1992) Plant Cell4:1575–1588); the zinc finger protein (Z) family (Klug and Schwabe(1995) FASEB J. 9: 597–604); the homeobox (HB) protein family (Buerglinin Guidebook to the Homeobox Genes, Duboule (ed.) (1994) OxfordUniversity Press); the CAAT-element binding proteins (Forsburg andGuarente (1989) Genes Dev. 3:1166–1178); the squamosa promoter bindingproteins (SPB) (Klein et al. (1996) Mol. Gen. Genet. 1996 250:7–16); theNAM protein family (Souer et al. (1996) Cell 85:159–170); the IAA/AUXproteins (Rouse et al. (1998) Science 279:1371–1373); the HLH/MYCprotein family (Littlewood et al. (1994) Prot. Profile 1:639–709); theDNA-binding protein (DBP) family (Tucker et al. (1994) EMBO J.13:2994–3002); the bZIP family of transcription factors (Foster et al.(1994) FASEB J. 8:192–200); the Box P-binding protein (the BPF-1) family(da Costa e Silva et al. (1993) Plant J. 4:125–135); the high mobilitygroup (HMG) family (Bustin and Reeves (1996) Prog. Nucl. Acids Res. Mol.Biol. 54:35–100); the scarecrow (SCR) family (Di Laurenzio et al. (1996)Cell 86:423–433); the GF14 family (Wu et al. (1997) Plant Physiol.114:1421–1431); the polycomb (PCOMB) family (Kennison (1995) Annu. Rev.Genet. 29:289–303); the teosinte branched (TEO) family (Luo et al.(1996) Nature 383:794–799; the ABI3 family (Giraudat et al. (1992) PlantCell 4:1251–1261); the triple helix (TH) family (Dehesh et al. (1990)Science 250:1397–1399); the EIL family (Chao et al. (1997) Cell89:1133–44); the AT-HOOK family (Reeves and Nissen (1990) J. Biol. Chem.265:8573–8582); the SIFA family (Zhou et al. (1995) Nucleic Acids Res.23:1165–1169); the bZIPT2 family (Lu and Ferl (1995) Plant Physiol.109:723); the YABBY family (Bowman et al. (1999) Development126:2387–96); the PAZ family (Bohmert et al. (1998) EMBO J. 17:170–80);a family of miscellaneous (MISC) transcription factors including theDPBF family (Kim et al. (1997) Plant J. 11:1237–1251) and the SPF1family (Ishiguro and Nakamura (1994) Mol. Gen. Genet. 244:563–571); thegolden (GLD) family (Hall et al. (1998) Plant Cell 10:925–936), theTUBBY family (Boggin et al, (1999) Science 286:2119–2125), the heatshock family (Wu C (1995) Annu Rev Cell Dev Biol 11:441–469), the ENBPfamily (Christiansen et al (1996) Plant Mol Biol 32:809–821), theRING-zinc family (Jensen et al. (1998) FEBS letters 436:283–287), thePDBP family (Janik et al Virology. (1989) 168:320–329), the PCF family(Cubas P, et al. Plant J. (1999) 18:215–22), the SRS (SHI-related)family (Fridborg et al Plant Cell (1999) 11:1019–1032), the CPP(cysteine-rich polycomb-like) family (Cvitanich et al Proc. Natl. Acad.Sci. USA (2000) 97:8163–8168), the ARF (auxin response factor) family(Ulmasov, et al. (1999) Proc. Natl. Acad. Sci. USA 96: 5844–5849), theSWI/SNF family (Collingwood et al J. Mol. End. 23:255–275), the ACBFfamily (Seguin et al (1997) Plant Mol Biol. 35:281–291), PCGL (CG-1like) family (da Costa e Silva et al. (1994) Plant Mol Biol. 25:921–924)the ARID family (Vazquez et al. (1999) Development. 126: 733–42), theJumonji family, Balciunas et al (2000, Trends Biochem Sci. 25: 274–276),the bZIP-NIN family (Schauser et al (1999) Nature 402: 191–195), the E2Ffamily Kaelin et al (1992) Cell 70: 351–364) and the GRF-like family(Knaap et al (2000) Plant Physiol. 122: 695–704). As indicated by anypart of the list above and as known in the art, transcription factorshave been sometimes categorized by class, family, and sub-familyaccording to their structural content and consensus DNA-binding sitemotif, for example. Many of the classes and many of the families andsub-families are listed here. However, the inclusion of one sub-familyand not another, or the inclusion of one family and not another, doesnot mean that the invention does not encompass polynucleotides orpolypeptides of a certain family or sub-family. The list provided hereis merely an example of the types of transcription factors and theknowledge available concerning the consensus sequences and consensusDNA-binding site motifs that help define them as known to those of skillin the art (each of the references noted above are specificallyincorporated herein by reference). A transcription factor may include,but is not limited to, any polypeptide that can activate or represstranscription of a single gene or a number of genes. This polypeptidegroup includes, but is not limited to, DNA-binding proteins, DNA-bindingprotein binding proteins, protein kinases, protein phosphatases,GTP-binding proteins, and receptors, and the like.

In addition to methods for modifying a plant phenotype by employing oneor more polynucleotides and polypeptides of the invention describedherein, the polynucleotides and polypeptides of the invention have avariety of additional uses. These uses include their use in therecombinant production (i.e., expression) of proteins; as regulators ofplant gene expression, as diagnostic probes for the presence ofcomplementary or partially complementary nucleic acids (including fordetection of natural coding nucleic acids); as substrates for furtherreactions, e.g., mutation reactions, PCR reactions, or the like; assubstrates for cloning e.g., including digestion or ligation reactions;and for identifying exogenous or endogenous modulators of thetranscription factors. A “polynucleotide” is a nucleic acid sequencecomprising a plurality of polymerized nucleotides, e.g., at least about15 consecutive polymerized nucleotides, optionally at least about 30consecutive nucleotides, at least about 50 consecutive nucleotides. Inmany instances, a polynucleotide comprises a nucleotide sequenceencoding a polypeptide (or protein) or a domain or fragment thereof.Additionally, the polynucleotide may comprise a promoter, an intron, anenhancer region, a polyadenylation site, a translation initiation site,5′ or 3′ untranslated regions, a reporter gene, a selectable marker, orthe like. The polynucleotide can be single stranded or double strandedDNA or RNA. The polynucleotide optionally comprises modified bases or amodified backbone. The polynucleotide can be, e.g., genomic DNA or RNA,a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, asynthetic DNA or RNA, or the like. The polynucleotide can comprise asequence in either sense or antisense orientations.

A “recombinant polynucleotide” is a polynucleotide that is not in itsnative state, e.g., the polynucleotide comprises a nucleotide sequencenot found in nature, or the polynucleotide is in a context other thanthat in which it is naturally found, e.g., separated from nucleotidesequences with which it typically is in proximity in nature, or adjacent(or contiguous with) nucleotide sequences with which it typically is notin proximity. For example, the sequence at issue can be cloned into avector, or otherwise recombined with one or more additional nucleicacid.

An “isolated polynucleotide” is a polynucleotide whether naturallyoccurring or recombinant, that is present outside the cell in which itis typically found in nature, whether purified or not. Optionally, anisolated polynucleotide is subject to one or more enrichment orpurification procedures, e.g., cell lysis, extraction, centrifugation,precipitation, or the like.

A “polypeptide” is an amino acid sequence comprising a plurality ofconsecutive polymerized amino acid residues e.g., at least about 15consecutive polymerized amino acid residues, optionally at least about30 consecutive polymerized amino acid residues, at least about 50consecutive polymerized amino acid residues. In many instances, apolypeptide comprises a polymerized amino acid residue sequence that isa transcription factor or a domain or portion or fragment thereof.Additionally, the polypeptide may comprise a localization domain, 2) anactivation domain, 3) a repression domain, 4) an oligomerization domainor 5) a DNA-binding domain, or the like. The polypeptide optionallycomprises modified amino acid residues, naturally occurring amino acidresidues not encoded by a codon, non-naturally occurring amino acidresidues.

A “recombinant polypeptide” is a polypeptide produced by translation ofa recombinant polynucleotide. A “synthetic polypeptide” is a polypeptidecreated by consecutive polymerization of isolated amino acid residuesusing methods well known in the art. An “isolated polypeptide,” whethera naturally occurring or a recombinant polypeptide, is more enriched in(or out of) a cell than the polypeptide in its natural state in a wildtype cell, e.g., more than about 5% enriched, more than about 10%enriched, or more than about 20%, or more than about 50%, or more,enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more,enriched relative to wild type standardized at 100%. Such an enrichmentis not the result of a natural response of a wild type plant.Alternatively, or additionally, the isolated polypeptide is separatedfrom other cellular components with which it is typically associated,e.g., by any of the various protein purification methods herein.

“Identity” or “similarity” refers to sequence similarity between twopolynucleotide sequences or between two polypeptide sequences, withidentity being a more strict comparison. The phrases “percent identity”and “% identity” refer to the percentage of sequence similarity found ina comparison of two or more polynucleotide sequences or two or morepolypeptide sequences. Identity or similarity can be determined bycomparing a position in each sequence that may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same nucleotide base or amino acid, then the molecules are identicalat that position. A degree of similarity or identity betweenpolynucleotide sequences is a function of the number of identical ormatching nucleotides at positions shared by the polynucleotidesequences. A degree of identity of polypeptide sequences is a functionof the number of identical amino acids at positions shared by thepolypeptide sequences. A degree of homology or similarity of polypeptidesequences is a function of the number of amino acids, i.e., structurallyrelated, at positions shared by the polypeptide sequences.

“Altered” nucleic acid sequences encoding polypeptide include thosesequences with deletions, insertions, or substitutions of differentnucleotides, resulting in a polynucleotide encoding a polypeptide withat least one functional characteristic of the polypeptide. Includedwithin this definition are polymorphisms that may or may not be readilydetectable using a particular oligonucleotide probe of thepolynucleotide encoding polypeptide, and improper or unexpectedhybridization to allelic variants, with a locus other than the normalchromosomal locus for the polynucleotide sequence encoding polypeptide.The encoded polypeptide protein may also be “altered”, and may containdeletions, insertions, or substitutions of amino acid residues thatproduce a silent change and result in a functionally equivalentpolypeptide. Deliberate amino acid substitutions may be made on thebasis of similarity in residue side chain chemistry, including, but notlimited to, polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological activity of polypeptide is retained. For example,negatively charged amino acids may include aspartic acid and glutamicacid, positively charged amino acids may include lysine and arginine,and amino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine; andphenylalanine and tyrosine. Alignments between different polypeptidesequences may be used to calculate “percentage sequence similarity”.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g., leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g., bracts, sepals, petals, stamens,carpels, anthers and ovules), seed (including embryo, endosperm, andseed coat) and fruit (the mature ovary), plant tissue (e.g., vasculartissue, ground tissue, and the like) and cells (e.g., guard cells, eggcells, and the like), and progeny of same. The class of plants that canbe used in the method of the invention is generally as broad as theclass of higher and lower plants amenable to transformation techniques,including angiosperms (monocotyledonous and dicotyledonous plants),gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, andmulticellular algae. (See for example, FIG. 1, adapted from Daly et al.2001 Plant Physiology 127:1328–1333; and see also Tudge, C., The Varietyof Life, Oxford University Press, New York, 2000, pp. 547–606.)

A “transgenic plant” refers to a plant that contains genetic materialnot found in a wild type plant of the same species, variety or cultivar.The genetic material may include a transgene, an insertional mutagenesisevent (such as by transposon or T-DNA insertional mutagenesis), anactivation tagging sequence, a mutated sequence, a homologousrecombination event or a sequence modified by chimeraplasty. Typically,the foreign genetic material has been introduced into the plant by humanmanipulation, but any method can be used as one of skill in the artrecognizes.

A transgenic plant may contain an expression vector or cassette. Theexpression cassette typically comprises a polypeptide-encoding sequenceoperably linked (i.e., under regulatory control of) to appropriateinducible or constitutive regulatory sequences that allow for theexpression of polypeptide. The expression cassette can be introducedinto a plant by transformation or by breeding after transformation of aparent plant. A plant refers to a whole plant as well as to a plantpart, such as seed, fruit, leaf, or root, plant tissue, plant cells orany other plant material, e.g., a plant explant, as well as to progenythereof, and to in vitro systems that mimic biochemical or cellularcomponents or processes in a cell.

“Ectopic expression or altered expression” in reference to apolynucleotide indicates that the pattern of expression in, e.g., atransgenic plant or plant tissue, is different from the expressionpattern in a wild type plant or a reference plant of the same species.The pattern of expression may also be compared with a referenceexpression pattern in a wild type plant of the same species. Forexample, the polynucleotide or polypeptide is expressed in a cell ortissue type other than a cell or tissue type in which the sequence isexpressed in the wild type plant, or by expression at a time other thanat the time the sequence is expressed in the wild type plant, or by aresponse to different inducible agents, such as hormones orenvironmental signals, or at different expression levels (either higheror lower) compared with those found in a wild type plant. The term alsorefers to altered expression patterns that are produced by lowering thelevels of expression to below the detection level or completelyabolishing expression. The resulting expression pattern can be transientor stable, constitutive or inducible. In reference to a polypeptide, theterm “ectopic expression or altered expression” further may relate toaltered activity levels resulting from the interactions of thepolypeptides with exogenous or endogenous modulators or frominteractions with factors or as a result of the chemical modification ofthe polypeptides.

A “fragment” or “domain,” with respect to a polypeptide, refers to asubsequence of the polypeptide. In some cases, the fragment or domain,is a subsequence of the polypeptide which performs at least onebiological function of the intact polypeptide in substantially the samemanner, or to a similar extent, as does the intact polypeptide. Forexample, a polypeptide fragment can comprise a recognizable structuralmotif or functional domain such as a DNA-binding site or domain thatbinds to a DNA promoter region, an activation domain, or a domain forprotein-protein interactions. Fragments can vary in size from as few as6 amino acids to the full length of the intact polypeptide, but arepreferably at least about 30 amino acids in length and more preferablyat least about 60 amino acids in length. In reference to apolynucleotide sequence, “a fragment” refers to any subsequence of apolynucleotide, typically, of at least about 15 consecutive nucleotides,preferably at least about 30 nucleotides, more preferably at least about50 nucleotides, of any of the sequences provided herein.

The invention also encompasses production of DNA sequences that encodetranscription factors and transcription factor derivatives, or fragmentsthereof, entirely by synthetic chemistry. After production, thesynthetic sequence may be inserted into any of the many availableexpression vectors and cell systems using reagents well known in theart. Moreover, synthetic chemistry may be used to introduce mutationsinto a sequence encoding transcription factors or any fragment thereof.

A “conserved domain”, with respect to a polypeptide, refers to a domainwithin a transcription factor family which exhibits a higher degree ofsequence homology, such as at least 65% sequence identity includingconservative substitutions, and preferably at least 80% sequenceidentity, and more preferably at least 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 90%, or atleast about 95%, or at least about 98% amino acid residue sequenceidentity of a polypeptide of consecutive amino acid residues. A fragmentor domain can be referred to as outside a consensus sequence or outsidea consensus DNA-binding site that is known to exist or that exists for aparticular transcription factor class, family, or sub-family. In thiscase, the fragment or domain will not include the exact amino acids of aconsensus sequence or consensus DNA-binding site of a transcriptionfactor class, family or sub-family, or the exact amino acids of aparticular transcription factor consensus sequence or consensusDNA-binding site. Furthermore, a particular fragment, region, or domainof a polypeptide, or a polynucleotide encoding a polypeptide, can be“outside a conserved domain” if all the amino acids of the fragment,region, or domain fall outside of a defined conserved domain(s) for apolypeptide or protein. The conserved domains for each of polypeptidesof SEQ ID NOs:2—2N, where N=2–123, are listed in Table 4 as described inExample VII. Also, many of the polypeptides of Table 4 have conserveddomains specifically indicated by start and stop sites. A comparison ofthe regions of the polypeptides in SEQ ID NOs:2—2N, where N 32 2–123, orof those in Table 4, allows one of skill in the art to identifyconserved domain(s) for any of the polypeptides listed or referred to inthis disclosure, including those in Table 4 or Table 5.

A “trait” refers to a physiological, morphological, biochemical, orphysical characteristic of a plant or particular plant material or cell.In some instances, this characteristic is visible to the human eye, suchas seed or plant size, or can be measured by biochemical techniques,such as detecting the protein, starch, or oil content of seed or leaves,or by observation of a metabolic or physiological process, e.g. bymeasuring uptake of carbon dioxide, or by the observation of theexpression level of a gene or genes, e.g., by employing Northernanalysis, RT-PCR, microarray gene expression assays, or reporter geneexpression systems, or by agricultural observations such as stresstolerance, yield, or pathogen tolerance. Any technique can be used tomeasure the amount of, comparative level of, or difference in anyselected chemical compound or macromolecule in the transgenic plants,however.

“Trait modification” refers to a detectable difference in acharacteristic in a plant ectopically expressing a polynucleotide orpolypeptide of the present invention relative to a plant not doing so,such as a wild type plant. In some cases, the trait modification can beevaluated quantitatively. For example, the trait modification can entailat least about a 2% increase or decrease in an observed trait(difference), at least a 5% difference, at least about a 10% difference,at least about a 20% difference, at least about a 30%, at least about a50%, at least about a 70%, or at least about a 100%, or an even greaterdifference compared with a wild type plant. It is known that there canbe a natural variation in the modified trait. Therefore, the traitmodification observed entails a change of the normal distribution of thetrait in the plants compared with the distribution observed in wild typeplant.

Traits Which may be Modified

Trait modifications of particular interest include those to seed (suchas embryo or endosperm), fruit, root, flower, leaf, stem, shoot,seedling or the like, including: enhanced tolerance to environmentalconditions including freezing, chilling, heat, drought, watersaturation, radiation and ozone; improved tolerance to microbial, fungalor viral diseases; improved tolerance to pest infestations, includingnematodes, mollicutes, parasitic higher plants or the like; decreasedherbicide sensitivity; improved tolerance of heavy metals or enhancedability to take up heavy metals; improved growth under poorphotoconditions (e.g., low light and/or short day length), or changes inexpression levels of genes of interest. Other phenotype that can bemodified relate to the production of plant metabolites, such asvariations in the production of taxol, tocopherol, tocotrienol, sterols,phytosterols, vitamins, wax monomers, anti-oxidants, amino acids,lignins, cellulose, tannins, prenyllipids (such as chlorophylls andcarotenoids), glucosinolates, and terpenoids, enhanced orcompositionally altered protein or oil production (especially in seeds),or modified sugar (insoluble or soluble) and/or starch composition.Physical plant characteristics that can be modified include celldevelopment (such as the number of trichomes), fruit and seed size andnumber, yields of plant parts such as stems, leaves, inflorescences, androots, the stability of the seeds during storage, characteristics of theseed pod (e.g., susceptibility to shattering), root hair length andquantity, internode distances, or the quality of seed coat. Plant growthcharacteristics that can be modified include growth rate, germinationrate of seeds, vigor of plants and seedlings, leaf and flowersenescence, male sterility, apomixis, flowering time, flower abscission,rate of nitrogen uptake, osmotic sensitivity to soluble sugarconcentrations, biomass or transpiration characteristics, as well asplant architecture characteristics such as apical dominance, branchingpatterns, number of organs, organ identity, organ shape or size.

Transcription Factors Modify Expression of Endogenous Genes

Expression of genes which encode transcription factors that modifyexpression of endogenous genes, polynucleotides, and proteins are wellknown in the art. In addition, transgenic plants comprising isolatedpolynucleotides encoding transcription factors may also modifyexpression of endogenous genes, polynucleotides, and proteins. Examplesinclude Peng et al. (1997, Genes and Development 11:3194–3205) and Penget al. (1999, Nature, 400:256–261). In addition, many others havedemonstrated that an Arabidopsis transcription factor expressed in anexogenous plant species elicits the same or very similar phenotypicresponse. See, for example, Fu et al. (2001, Plant Cell 13:1791–1802);Nandi et al. (2000, Curr. Biol. 10:215–218); Coupland (1995, Nature377:482–483); and Weigel and Nilsson (1995, Nature 377:482–500).

In another example, Mandel et al. (1992, Cell 71-133–143) and Suzuki etal. (2001, Plant J. 28:409–418) teach that a transcription factorexpressed in another plant species elicits the same or very similarphenotypic response of the endogenous sequence, as often predicted inearlier studies of Arabidopsis transcription factors in Arabidopsis (seeMandel et al., 1992, supra; Suzuki et al., 2001, supra).

Other examples include Müller et al. (2001, Plant J. 28:169–179); Kim etal. (2001, Plant J. 25:247–259); Kyozuka and Shimamoto (2002, Plant CellPhysiol. 43:130–135); Boss and Thomas (2002, Nature, 416:847–850); He etal. (2000, Transgenic Res., 9:223–227); and Robson et al. (2001, PlantJ. 28:619–631).

In yet another example, Gilmour et al. (1998, Plant J. 16:433–442) teachan Arabidopsis AP2 transcription factor, CBF1, which, when overexpressedin transgenic plants, increases plant freezing tolerance. Jaglo et al(2001, Plant Physiol. 127:910–017) further identified sequences inBrassica napus which encode CBF-like genes and that transcripts forthese genes accumulated rapidly in response to low temperature.Transcripts encoding CBF-like proteins were also found to accumulaterapidly in response to low temperature in wheat, as well as in tomato.An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye,and tomato revealed the presence of conserved amino acid sequences,PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA bindingdomains of the proteins and distinguish them from other members of theAP2/EREBP protein family. (See Jaglo et al., supra.)

Polypeptides and Polynucleotides of the Invention

The present invention provides, among other things, transcriptionfactors (TFs), and transcription factor homologue polypeptides, andisolated or recombinant polynucleotides encoding the polypeptides, ornovel variant polypeptides or polynucleotides encoding novel variants oftranscription factors derived from the specific sequences provided here.These polypeptides and polynucleotides may be employed to modify aplant's characteristic.

Exemplary polynucleotides encoding the polypeptides of the inventionwere identified in the Arabidopsis thaliana GenBank database usingpublicly available sequence analysis programs and parameters. Sequencesinitially identified were then further characterized to identifysequences comprising specified sequence strings corresponding tosequence motifs present in families of known transcription factors. Inaddition, further exemplary polynucleotides encoding the polypeptides ofthe invention were identified in the plant GenBank database usingpublicly available sequence analysis programs and parameters. Sequencesinitially identified were then further characterized to identifysequences comprising specified sequence strings corresponding tosequence motifs present in families of known transcription factors.Polynucleotide sequences meeting such criteria were confirmed astranscription factors.

Additional polynucleotides of the invention were identified by screeningArabidopsis thaliana and/or other plant cDNA libraries with probescorresponding to known transcription factors under low stringencyhybridization conditions. Additional sequences, including full lengthcoding sequences were subsequently recovered by the rapid amplificationof cDNA ends (RACE) procedure, using a commercially available kitaccording to the manufacturer's instructions. Where necessary, multiplerounds of RACE are performed to isolate 5′ and 3′ ends. The full lengthcDNA was then recovered by a routine end-to-end polymerase chainreaction (PCR) using primers specific to the isolated 5′ and 3′ ends.Exemplary sequences are provided in the Sequence Listing.

The polynucleotides of the invention can be or were ectopicallyexpressed in overexpressor or knockout plants and the changes in thecharacteristic(s) or trait(s) of the plants observed. Therefore, thepolynucleotides and polypeptides can be employed to improve thecharacteristics of plants.

The polynucleotides of the invention can be or were ectopicallyexpressed in overexpressor plant cells and the changes in the expressionlevels of a number of genes, polynucleotides, and/or proteins of theplant cells observed. Therefore, the polynucleotides and polypeptidescan be employed to change expression levels of a genes, polynucleotides,and/or proteins of plants.

Producing Polypeptides

The polynucleotides of the invention include sequences that encodetranscription factors and transcription factor homologue polypeptidesand sequences complementary thereto, as well as unique fragments ofcoding sequence, or sequence complementary thereto. Such polynucleotidescan be, e.g., DNA or RNA, e.g., mRNA, cRNA, synthetic RNA, genomic DNA,cDNA synthetic DNA, oligonucleotides, etc. The polynucleotides areeither double-stranded or single-stranded, and include either, or bothsense (i.e., coding) sequences and antisense (i.e., non-coding,complementary) sequences. The polynucleotides include the codingsequence of a transcription factor, or transcription factor homologuepolypeptide, in isolation, in combination with additional codingsequences (e.g., a purification tag, a localization signal, as afusion-protein, as a pre-protein, or the like), in combination withnon-coding sequences (e.g., introns or inteins, regulatory elements suchas promoters, enhancers, terminators, and the like), and/or in a vectoror host environment in which the polynucleotide encoding a transcriptionfactor or transcription factor homologue polypeptide is an endogenous orexogenous gene.

A variety of methods exist for producing the polynucleotides of theinvention. Procedures for identifying and isolating DNA clones are wellknown to those of skill in the art, and are described in, e.g., Bergerand Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (“Berger”); Sambrooket al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1–3, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”)and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (supplemented through 2000)(“Ausubel”).

Alternatively, polynucleotides of the invention, can be produced by avariety of in vitro amplification methods adapted to the presentinvention by appropriate selection of specific or degenerate primers.Examples of protocols sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR) the ligase chain reaction (LCR), Qbeta-replicase amplification andother RNA polymerase mediated techniques (e.g., NASBA), e.g., for theproduction of the homologous nucleic acids of the invention are found inBerger (supra), Sambrook (supra), and Ausubel (supra), as well as Mulliset al., (1987) PCR Protocols A Guide to Methods and Applications (Inniset al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis).Improved methods for cloning in vitro amplified nucleic acids aredescribed in Wallace et al., U.S. Pat. No. 5,426,039. Improved methodsfor amplifying large nucleic acids by PCR are summarized in Cheng et al.(1994) Nature 369: 684–685 and the references cited therein, in whichPCR amplicons of up to 40kb are generated. One of skill will appreciatethat essentially any RNA can be converted into a double stranded DNAsuitable for restriction digestion, PCR expansion and sequencing usingreverse transcriptase and a polymerase. See, e.g., Ausubel, Sambrook andBerger, all supra.

Alternatively, polynucleotides and oligonucleotides of the invention canbe assembled from fragments produced by solid-phase synthesis methods.Typically, fragments of up to approximately 100 bases are individuallysynthesized and then enzymatically or chemically ligated to produce adesired sequence, e.g., a polynucleotide encoding all or part of atranscription factor. For example, chemical synthesis using thephosphoramidite method is described, e.g., by Beaucage et al. (1981)Tetrahedron Letters 22:1859–1869; and Matthes et al. (1984) EMBO J.3:801–805. According to such methods, oligonucleotides are synthesized,purified, annealed to their complementary strand, ligated and thenoptionally cloned into suitable vectors. And if so desired, thepolynucleotides and polypeptides of the invention can be custom orderedfrom any of a number of commercial suppliers.

Homologous Sequences

Sequences homologous, i.e., that share significant sequence identity orsimilarity, to those provided in the Sequence Listing, derived fromArabidopsis thaliana or from other plants of choice are also an aspectof the invention. Homologous sequences can be derived from any plantincluding monocots and dicots and in particular agriculturally importantplant species, including but not limited to, crops such as soybean,wheat, corn, potato, cotton, rice, rape, oilseed rape (includingcanola), sunflower, alfalfa, sugarcane and turf; or fruits andvegetables, such as banana, blackberry, blueberry, strawberry, andraspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant,grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers,pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato,watermelon, rosaceous fruits (such as apple, peach, pear, cherry andplum) and vegetable brassicas (such as broccoli, cabbage, cauliflower,Brussels sprouts, and kohlrabi). Other crops, fruits and vegetableswhose phenotype can be changed include barley, rye, millet, sorghum,currant, avocado, citrus fruits such as oranges, lemons, grapefruit andtangerines, artichoke, cherries, nuts such as the walnut and peanut,endive, leek, roots, such as arrowroot, beet, cassava, turnip, radish,yam, and sweet potato, and beans. The homologous sequences may also bederived from woody species, such pine, poplar and eucalyptus, or mint orother labiates.

Orthologs and Paralogs

Several different methods are known by those of skill in the art foridentifying and defining these functionally homologous sequences. Threegeneral methods for defining paralogs and orthologs are described; aparalog or ortholog or homolog may be identified by one or more of themethods described below.

Orthologs and paralogs are evolutionarily related genes that havesimilar sequence and similar functions. Orthologs are structurallyrelated genes in different species that are derived by a speciationevent. Paralogs are structurally related genes within a single speciesand that are derived by a duplication event.

Within a single plant species, gene duplication may cause two copies ofa particular gene, giving rise to two or more genes with similarsequence and similar function known as paralogs. A paralog is thereforea similar gene with a similar function within the same species. Paralogstypically cluster together or in the same lade (a group of similargenes) when a gene family phylogeny is analyzed using programs such asCLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22:4673–4680; Higginset al. (1996) Methods Enzymol. 266 383–402). Groups of similar genes canalso be identified with pair-wise BLAST analysis (Feng and Doolittle(1987) J. Mol. Evol. 25:351–360). For example, a clade of very similarMADS domain transcription factors from Arabidopsis all share a commonfunction in flowering time (Ratcliffe et al. (2001) Plant Physiol.126:122–132), and a group of very similar AP2 domain transcriptionfactors from Arabidopsis are involved in tolerance of plants to freezing(Gilmour et al. (1998) Plant J. 16:433–442). Analysis of groups ofsimilar genes with similar function that fall within one clade can yieldsub-sequences that are particular to the clade. These sub-sequences,known as consensus sequences, can not only be used to define thesequences within each lade, but define the functions of these genes;genes within a lade may contain paralogous sequences, or orthologoussequences that share the same function. (See also, for example, Mount,D. W. (2001) Bioinformatics: Sequence and Genome Analysis Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. page 543.)

Speciation, the production of new species from a parental species, canalso give rise to two or more genes with similar sequence and similarfunction. These genes, termed orthologs, often have an identicalfunction within their host plants and are often interchangeable betweenspecies without losing function. Because plants have common ancestors,many genes in any plant species will have a corresponding orthologousgene in another plant species. Once a phylogenic tree for a gene familyof one species has been constructed using a program such as CLUSTAL(Thompson et al. (1994) Nucleic Acids Res. 22:4673–4680; Higgins et al.(1996) Methods Enzymol. 266:383–402), potential orthologous sequencescan placed into the phylogenetic tree and its relationship to genes fromthe species of interest can be determined. Once the ortholog pair hasbeen identified, the function of the test ortholog can be determined bydetermining the function of the reference ortholog.

Transcription factors that are homologous to the listed sequences willtypically share at least about 30% amino acid sequence identity, or atleast about 30% amino acid sequence identity outside of a knownconsensus sequence or consensus DNA-binding site. More closely relatedtranscription factors can share at least about 50%, about 60%, about65%, about 70%, about 75% or about 80% or about 90% or about 95% orabout 98% or more sequence identity with the listed sequences, or withthe listed sequences but excluding or outside a known consensus sequenceor consensus DNA-binding site, or with the listed sequences excludingone or all conserved domain. Factors that are most closely related tothe listed sequences share, e.g., at least about 85%, about 90% or about95% or more % sequence identity to the listed sequences, or to thelisted sequences but excluding or outside a known consensus sequence orconsensus DNA-binding site or outside one or all conserved domain. Atthe nucleotide level, the sequences will typically share at least about40% nucleotide sequence identity, preferably at least about 50%, about60%, about 70% or about 80% sequence identity, and more preferably about85%, about 90%, about 95% or about 97% or more sequence identity to oneor more of the listed sequences, or to a listed sequence but excludingor outside a known consensus sequence or consensus DNA-binding site, oroutside one or all conserved domain. The degeneracy of the genetic codeenables major variations in the nucleotide sequence of a polynucleotidewhile maintaining the amino acid sequence of the encoded protein.Conserved domains within a transcription factor family may exhibit ahigher degree of sequence homology, such as at least 65% sequenceidentity including conservative substitutions, and preferably at least80% sequence identity, and more preferably at least 85%, or at leastabout 86%, or at least about 87%, or at least about 88%, or at leastabout 90%, or at least about 95%, or at least about 98% sequenceidentity. Transcription factors that are homologous to the listedsequences should share at least 30%, or at least about 60%, or at leastabout 75%, or at least about 80%, or at least about 90%, or at leastabout 95% amino acid sequence identity over the entire length of thepolypeptide or the homolog. In addition, transcription factors that arehomologous to the listed sequences should share at least 30%, or atleast about 60%, or at least about 75%, or at least about 80%, or atleast about 90%, or at least about 95% amino acid sequence similarityover the entire length of the polypeptide or the homolog.

Percent identity can be determined electronically, e.g., by using theMEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program cancreate alignments between two or more sequences according to differentmethods, e.g., the clustal method. (See, e.g., Higgins, D. G. and P. M.Sharp (1988) Gene 73:237–244.) The clustal algorithm groups sequencesinto clusters by examining the distances between all pairs. The clustersare aligned pairwise and then in groups. Other alignment algorithms orprograms may be used, including FASTA, BLAST, or ENTREZ, FASTA andBLAST. These are available as a part of the GCG sequence analysispackage (University of Wisconsin, Madison, Wis.), and can be used withor without default settings. ENTREZ is available through the NationalCenter for Biotechnology Information. In one embodiment, the percentidentity of two sequences can be determined by the GCG program with agap weight of 1, e.g., each amino acid gap is weighted as if it were asingle amino acid or nucleotide mismatch between the two sequences (seeU.S. Pat. No. 6,262,333).

Other techniques for alignment are described in Methods in Enzymology,vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996),ed. Doolittle, Academic Press, Inc., San Diego, Calif., USA. Preferably,an alignment program that permits gaps in the sequence is utilized toalign the sequences. The Smith-Waterman is one type of algorithm thatpermits gaps in sequence alignments. See Methods Mol. Biol. 70: 173–187(1997). Also, the GAP program using the Needleman and Wunsch alignmentmethod can be utilized to align sequences. An alternative searchstrategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCHuses a Smith-Waterman algorithm to score sequences on a massivelyparallel computer. This approach improves ability to pick up distantlyrelated matches, and is especially tolerant of small gaps and nucleotidesequence errors. Nucleic acid-encoded amino acid sequences can be usedto search both protein and DNA databases.

The percentage similarity between two polypeptide sequences, e.g.,sequence A and sequence B, is calculated by dividing the length ofsequence A, minus the number of gap residues in sequence A, minus thenumber of gap residues in sequence B, into the sum of the residuematches between sequence A and sequence B, times one hundred. Gaps oflow or of no similarity between the two amino acid sequences are notincluded in determining percentage similarity. Percent identity betweenpolynucleotide sequences can also be counted or calculated by othermethods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein,J. (1990) Methods Enzymol. 183:626–645.) Identity between sequences canalso be determined by other methods known in the art, e.g., by varyinghybridization conditions (see U.S. Patent Application No. 20010010913).

Thus, the invention provides methods for identifying a sequence similaror paralogous or orthologous or homologous to one or morepolynucleotides as noted herein, or one or more target polypeptidesencoded by the polynucleotides, or otherwise noted herein and mayinclude linking or associating a given plant phenotype or gene functionwith a sequence. In the methods, a sequence database is provided(locally or across an inter or intra net) and a query is made againstthe sequence database using the relevant sequences herein and associatedplant phenotypes or gene functions.

In addition, one or more polynucleotide sequences or one or morepolypeptides encoded by the polynucleotide sequences may be used tosearch against a BLOCKS (Bairoch et al. (1997) Nucleic Acids Res.25:217–221), PFAM, and other databases which contain previouslyidentified and annotated motifs, sequences and gene functions. Methodsthat search for primary sequence patterns with secondary structure gappenalties (Smith et al. (1992) Protein Engineering 5:35–51) as well asalgorithms such as Basic Local Alignment Search Tool (BLAST; Altschul,S. F. (1993) J. Mol. Evol. 36:290–300; Altschul et al. (1990) supra),BLOCKS (Henikoff, S. and Henikoff, G. J. (1991) Nucleic Acids Research19:6565–6572), Hidden Markov Models (HMM; Eddy, S. R. (1996) Cur. Opin.Str. Biol. 6:361–365; Sonnhammer et al. (1997) Proteins 28:405–420), andthe like, can be used to manipulate and analyze polynucleotide andpolypeptide sequences encoded by polynucleotides. These databases,algorithms and other methods are well known in the art and are describedin Ausubel et al. (1997; Short Protocols in Molecular Biology, JohnWiley & Sons, New York N.Y., unit 7.7) and in Meyers, R. A. (1995;Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., p856–853).

Furthermore, methods using manual alignment of sequences similar orhomologous to one or more polynucleotide sequences or one or morepolypeptides encoded by the polynucleotide sequences may be used toidentify regions of similarity and conserved domains. Such manualmethods are well-known of those of skill in the art and can include, forexample, comparisons of tertiary structure between a polypeptidesequence encoded by a polynucleotide which comprises a known functionwith a polypeptide sequence encoded by a polynucleotide sequence whichhas a function not yet determined. Such examples of tertiary structuremay comprise predicted alpha helices, beta-sheets, amphipathic helices,leucine zipper motifs, zinc finger motifs, proline-rich regions,cysteine repeat motifs, and the like.

Identifying Polynucleotides or Nucleic Acids by Hybridization

Polynucleotides homologous to the sequences illustrated in the SequenceListing and tables can be identified, e.g., by hybridization to eachother under stringent or under highly stringent conditions. Singlestranded polynucleotides hybridize when they associate based on avariety of well characterized physical-chemical forces, such as hydrogenbonding, solvent exclusion, base stacking and the like. The stringencyof a hybridization reflects the degree of sequence identity of thenucleic acids involved, such that the higher the stringency, the moresimilar are the two polynucleotide strands. Stringency is influenced bya variety of factors, including temperature, salt concentration andcomposition, organic and non-organic additives, solvents, etc. presentin both the hybridization and wash solutions and incubations (and numberthereof), as described in more detail in the references cited above.Encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NOs: 26; 46; 176; 114; 142; 144;82; 50; 72; 96; 18; 22; 24; and 240, and fragments thereof under variousconditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger(1987) Methods Enzymol. 152:399–407; Kimmel, A. R. (1987) MethodsEnzymol. 152:507–511.) Estimates of homology are provided by eitherDNA-DNA or DNA-RNA hybridization under conditions of stringency as iswell understood by those skilled in the art (Haames and Higgins, Eds.(1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringencyconditions can be adjusted to screen for moderately similar fragments,such as homologous sequences from distantly related organisms, to highlysimilar fragments, such as genes that duplicate functional enzymes fromclosely related organisms. Post-hybridization washes determinestringency conditions.

In addition to the nucleotide sequences listed in Tables 4 and 5, fulllength cDNA, orthologs, paralogs and homologs of the present nucleotidesequences may be identified and isolated using well known methods. ThecDNA libraries orthologs, paralogs and homologs of the presentnucleotide sequences may be screened using hybridization methods todetermine their utility as hybridization target or amplification probes.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is about 5° C. to20° C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Nucleic acidmolecules that hybridize under stringent conditions will typicallyhybridize to a probe based on either the entire cDNA or selectedportions, e.g., to a unique subsequence, of the cDNA under washconditions of 0.2×SSC to 2.0×SSC, 0.1% SDS at 50–65° C. For example,high stringency is about 0.2×SSC, 0.1% SDS at 65° C. Ultra-highstringency will be the same conditions except the wash temperature israised about 3 to about 5° C., and ultra-ultra-high stringency will bethe same conditions except the wash temperature is raised about 6 toabout 9° C. For identification of less closely related homologues washescan be performed at a lower temperature, e.g., 50° C. In general,stringency is increased by raising the wash temperature and/ordecreasing the concentration of SSC, as known in the art.

In another example, stringent salt concentration will ordinarily be lessthan about 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and most preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and most preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μ/ml ssDNA. Useful variations onthese conditions will be readily apparent to those skilled in the art.

The washing steps that follow hybridization can also vary in stringency.Wash stringency conditions can be defined by salt concentration and bytemperature. As above, wash stringency can be increased by decreasingsalt concentration or by increasing temperature. For example, stringentsalt concentration for the wash steps will preferably be less than about30 mM NaCl and 3 mM trisodium citrate, and most preferably less thanabout 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperatureconditions for the wash steps will ordinarily include temperature of atleast about 25° C., more preferably of at least about 42° C. Anotherpreferred set of highly stringent conditions uses two final washes in0.1×SSC, 0.1% SDS at 65° C. The most preferred high stringency washesare of at least about 68° C. For example, in a preferred embodiment,wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate,and 0.1% SDS. In a more preferred embodiment, wash steps will occur at42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a mostpreferred embodiment, the wash steps will occur at 68° C. in 15 mM NaCl,1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on theseconditions will be readily apparent to those skilled in the art (seeU.S. Patent Application No. 20010010913).

As another example, stringent conditions can be selected such that anoligonucleotide that is perfectly complementary to the codingoligonucleotide hybridizes to the coding oligonucleotide with at leastabout a 5–10× higher signal to noise ratio than the ratio forhybridization of the perfectly complementary oligonucleotide to anucleic acid encoding a transcription factor known as of the filing dateof the application. Conditions can be selected such that a higher signalto noise ratio is observed in the particular assay which is used, e.g.,about 15×, 25×, 35×, 50× or more. Accordingly, the subject nucleic acidhybridizes to the unique coding oligonucleotide with at least a 2×higher signal to noise ratio as compared to hybridization of the codingoligonucleotide to a nucleic acid encoding known polypeptide. Again,higher signal to noise ratios can be selected, e.g., about 5×, 10×, 25×,35×, 50× or more. The particular signal will depend on the label used inthe relevant assay, e.g., a fluorescent label, a colorimetric label, aradioactive label, or the like.

Alternatively, transcription factor homolog polypeptides can be obtainedby screening an expression library using antibodies specific for one ormore transcription factors. With the provision herein of the disclosedtranscription factor, and transcription factor homologue nucleic acidsequences, the encoded polypeptide(s) can be expressed and purified in aheterologous expression system (e.g., E. coli) and used to raiseantibodies (monoclonal or polyclonal) specific for the polypeptide(s) inquestion. Antibodies can also be raised against synthetic peptidesderived from transcription factor, or transcription factor homologue,amino acid sequences. Methods of raising antibodies are well known inthe art and are described in Harlow and Lane (1988) Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York. Suchantibodies can then be used to screen an expression library producedfrom the plant from which it is desired to clone additionaltranscription factor homologues, using the methods described above. Theselected cDNAs can be confirmed by sequencing and enzymatic activity.

Sequence Variations

It will readily be appreciated by those of skill in the art, that any ofa variety of polynucleotide sequences are capable of encoding thetranscription factors and transcription factor homologue polypeptides ofthe invention. Due to the degeneracy of the genetic code, many diffidentpolynucleotides can encode identical and/or substantially similarpolypeptides in addition to those sequences illustrated in the SequenceListing. Nucleic acids having a sequence that differs from the sequencesshown in the Sequence Listing, or complementary sequences, that encodefunctionally equivalent peptides (i.e., peptides having some degree ofequivalent or similar biological activity) but differ in sequence fromthe sequence shown in the sequence listing due to degeneracy in thegenetic code, are also within the scope of the invention.

Altered polynucleotide sequences encoding polypeptides include thosesequences with deletions, insertions, or substitutions of differentnucleotides, resulting in a polynucleotide encoding a polypeptide withat least one functional characteristic of the instant polypeptides.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding the instant polypeptides, and improper orunexpected hybridization to allelic variants, with a locus other thanthe normal chromosomal locus for the polynucleotide sequence encodingthe instant polypeptides.

Allelic variant refers to any of two or more alternative forms of a geneoccupying the same chromosomal locus. Allelic variation arises naturallythrough mutation, and may result in phenotypic polymorphism withinpopulations. Gene mutations can be silent (i.e., no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequence. The term allelic variant is also used herein to denote aprotein encoded by an allelic variant of a gene. Splice variant refersto alternative forms of RNA transcribed from a gene. Splice variationarises naturally through use of alternative splicing sites within atranscribed RNA molecule, or less commonly between separatelytranscribed RNA molecules, and may result in several mRNAs transcribedfrom the same gene. Splice variants may encode polypeptides havingaltered amino acid sequence. The term splice variant is also used hereinto denote a protein encoded by a splice variant of an mRNA transcribedfrom a gene.

Those skilled in the art would recognize that G481, SEQ ID NO: 114,represents a single transcription factor; allelic variation andalternative splicing may be expected to occur. Allelic variants of SEQID NO: 113 can be cloned by probing cDNA or genomic libraries fromdifferent individual organisms according to standard procedures. Allelicvariants of the DNA sequence shown in SEQ ID NO:113, including thosecontaining silent mutations and those in which mutations result in aminoacid sequence changes, are within the scope of the present invention, asare proteins which are allelic variants of SEQ ID NO: 114. cDNAsgenerated from alternatively spliced mRNAs, which retain the propertiesof the transcription factor are included within the scope of the presentinvention, as are polypeptides encoded by such cDNAs and mRNAs. Allelicvariants and splice variants of these sequences can be cloned by probingcDNA or genomic libraries from different individual organisms or tissuesaccording to standard procedures known in the art (see U.S. Pat. No.6,388,064).

For example, Table 1 illustrates, e.g., that the codons AGC, AGT, TCA,TCC, TCG, and TCT all encode the same amino acid: serine. Accordingly,at each position in the sequence where there is a codon encoding serine,any of the above trinucleotide sequences can be used without alteringthe encoded polypeptide.

TABLE 1 Amino acid Possible Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C TGC TGT Aspartic acid Asp D GAC GAT Glutamic acid Glu EGAA GAG Phenylalanine Phe F TTC TTT Glycine Gly G GGA GGC GGG GGTHistidine His H CAC CAT Isoleucine Ile I ATA ATC ATT Lysine Lys K AAAAAG Leucine Leu L TTA TTG CTA CTC CTG CTT Methionine Met M ATGAsparagine Asn N AAC AAT Proline Pro P CCA CCC CCG CCT Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGT Serine Ser S AGC AGT TCATCC TCG TCT Threonine Thr T ACA ACC ACG ACT Valine Val V GTA GTC GTG GTTTryptophan Trp W TGG Tyrosine Tyr Y TAC TAT

Sequence alterations that do not change the amino acid sequence encodedby the polynucleotide are termed “silent” variations. With the exceptionof the codons ATG and TGG, encoding methionine and tryptophan,respectively, any of the possible codons for the same amino acid can besubstituted by a variety of techniques, e.g., site-directed mutagenesis,available in the art. Accordingly, any and all such variations of asequence selected from the above table are a feature of the invention.

In addition to silent variations, other conservative variations thatalter one, or a few amino acids in the encoded polypeptide, can be madewithout altering the function of the polypeptide, these conservativevariants are, likewise, a feature of the invention.

For example, substitutions, deletions and insertions introduced into thesequences provided in the Sequence Listing are also envisioned by theinvention. Such sequence modifications can be engineered into a sequenceby site-directed mutagenesis (Wu (ed.) Meth. Enzymol. (1993) vol. 217,Academic Press) or the other methods noted below. Amino acidsubstitutions are typically of single residues; insertions usually willbe on the order of about from 1 to 10 amino acid residues; and deletionswill range about from 1 to 30 residues. In preferred embodiments,deletions or insertions are made in adjacent pairs, e.g., a deletion oftwo residues or insertion of two residues. Substitutions, deletions,insertions or any combination thereof can be combined to arrive at asequence. The mutations that are made in the polynucleotide encoding thetranscription factor should not place the sequence out of reading frameand should not create complementary regions that could produce secondarymRNA structure. Preferably, the polypeptide encoded by the DNA performsthe desired function.

Conservative substitutions are those in which at least one residue inthe amino acid sequence has been removed and a different residueinserted in its place. Such substitutions generally are made inaccordance with the Table 2 when it is desired to maintain the activityof the protein. Table 2 shows amino acids which can be substituted foran amino acid in a protein and which are typically regarded asconservative substitutions.

TABLE 2 Conservative Residue Substitutions Ala Ser Arg Lys Asn Gln; HisAsp Glu Gln Asn Cys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val LeuIle; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly ThrSer; Val Trp Tyr Tyr Trp; Phe Val Ile; Leu

Similar substitutions are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Such substitutions generally are made in accordance with theTable 3 when it is desired to maintain the activity of the protein.Table 3 shows amino acids which can be substituted for an amino acid ina protein and which are typically regarded as structural and functionalsubstitutions. For example, a residue in column 1 of Table 3 may besubstituted with residue in column 2; in addition, a residue in column 2of Table 3 may be substituted with the residue of column 1.

TABLE 3 Residue Similar Substitutions Ala Ser; Thr; Gly; Val; Leu; IleArg Lys; His; Gly Asn Gln; His; Gly; Ser; Thr Asp Glu, Ser; Thr Gln Asn;Ala Cys Ser; Gly Glu Asp Gly Pro; Arg His Asn; Gln; Tyr; Phe; Lys; ArgIle Ala; Leu; Val; Gly; Met Leu Ala; Ile; Val; Gly; Met Lys Arg; His;Gln; Gly; Pro Met Leu; Ile; Phe Phe Met; Leu; Tyr; Trp; His; Val; AlaSer Thr; Gly; Asp; Ala; Val; Ile; His Thr Ser; Val; Ala; Gly Trp Tyr;Phe; His Tyr Trp; Phe; His Val Ala; Ile; Leu; Gly; Thr; Ser; Glu

Substitutions that are less conservative than those in Table 2 can beselected by picking residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in proteinproperties will be those in which (a) a hydrophilic residue, e.g., serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d)a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine.

Further Modifying Sequences of the Invention—Mutation/Forced Evolution

In addition to generating silent or conservative substitutions as noted,above, the present invention optionally includes methods of modifyingthe sequences of the Sequence Listing. In the methods, nucleic acid orprotein modification methods are used to alter the given sequences toproduce new sequences and/or to chemically or enzymatically modify givensequences to change the properties of the nucleic acids or proteins.

Thus, in one embodiment, given nucleic acid sequences are modified,e.g., according to standard mutagenesis or artificial evolution methodsto produce modified sequences. The modified sequences may be createdusing purified natural polynucleotides isolated from any organism or maybe synthesized from purified compositions and chemicals using chemicalmeans well know to those of skill in the art. For example, Ausubel,supra, provides additional details on mutagenesis methods. Artificialforced evolution methods are described, for example, by Stemmer (1994)Nature 370:389–391, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747–10751, and U.S. Pat. Nos. 5,811,238, 5,837,500, and 6,242,568.Methods for engineering synthetic transcription factors and otherpolypeptides are described, for example, by Zhang et al. (2000) J. Biol.Chem. 275:33850–33860, Liu et al. (2001) J. Biol. Chem. 276:11323–11334,and Isalan et al. (2001) Nature Biotechnol. 19:656–660. Many othermutation and evolution methods are also available and expected to bewithin the skill of the practitioner.

Similarly, chemical or enzymatic alteration of expressed nucleic acidsand polypeptides can be performed by standard methods. For example,sequence can be modified by addition of lipids, sugars, peptides,organic or inorganic compounds, by the inclusion of modified nucleotidesor amino acids, or the like. For example, protein modificationtechniques are illustrated in Ausubel, supra. Further details onchemical and enzymatic modifications can be found herein. Thesemodification methods can be used to modify any given sequence, or tomodify any sequence produced by the various mutation and artificialevolution modification methods noted herein.

Accordingly, the invention provides for modification of any givennucleic acid by mutation, evolution, chemical or enzymatic modification,or other available methods, as well as for the products produced bypracticing such methods, e.g., using the sequences herein as a startingsubstrate for the various modification approaches.

For example, optimized coding sequence containing codons preferred by aparticular prokaryotic or eukaryotic host can be used e.g., to increasethe rate of translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced using a non-optimized sequence. Translation stopcodons can also be modified to reflect host preference. For example,preferred stop codons for Saccharomyces cerevisiae and mammals are TAAand TGA, respectively. The preferred stop codon for monocotyledonousplants is TGA, whereas insects and E. coli prefer to use TAA as the stopcodon.

The polynucleotide sequences of the present invention can also beengineered in order to alter a coding sequence for a variety of reasons,including but not limited to, alterations which modify the sequence tofacilitate cloning, processing and/or expression of the gene product.For example, alterations are optionally introduced using techniqueswhich are well known in the art, e.g., site-directed mutagenesis, toinsert new restriction sites, to alter glycosylation patterns, to changecodon preference, to introduce splice sites, etc.

Furthermore, a fragment or domain derived from any of the polypeptidesof the invention can be combined with domains derived from othertranscription factors or synthetic domains to modify the biologicalactivity of a transcription factor. For instance, a DNA-binding domainderived from a transcription factor of the invention can be combinedwith the activation domain of another transcription factor or with asynthetic activation domain. A transcription activation domain assistsin initiating transcription from a DNA-binding site. Examples includethe transcription activation region of VP16 or GAL4 (Moore et al. (1998)Proc. Natl. Acad. Sci. USA 95: 376–381; and Aoyama et al. (1995) PlantCell 7:1773–1785), peptides derived from bacterial sequences (Ma andPtashne (1987) Cell 51; 113–119) and synthetic peptides (Giniger andPtashne, (1987) Nature 330:670–672).

Expression and Modification of Polypeptides

Typically, polynucleotide sequences of the invention are incorporatedinto recombinant DNA (or RNA) molecules that direct expression ofpolypeptides of the invention in appropriate host cells, transgenicplants, in vitro translation systems, or the like. Due to the inherentdegeneracy of the genetic code, nucleic acid sequences which encodesubstantially the same or a functionally equivalent amino acid sequencecan be substituted for any listed sequence to provide for cloning andexpressing the relevant homologue.

Vectors, Promoters, and Expression Systems

The present invention includes recombinant constructs comprising one ormore of the nucleic acid sequences herein. The constructs typicallycomprise a vector, such as a plasmid, a cosmid, a phage, a virus (e.g.,a plant virus), a bacterial artificial chromosome (BAC), a yeastartificial chromosome (YAC), or the like, into which a nucleic acidsequence of the invention has been inserted, in a forward or reverseorientation. In a preferred aspect of this embodiment, the constructfurther comprises regulatory sequences, including, for example, apromoter, operably linked to the sequence. Large numbers of suitablevectors and promoters are known to those of skill in the art, and arecommercially available.

General texts that describe molecular biological techniques usefulherein, including the use and production of vectors, promoters and manyother relevant topics, include Berger, Sambrook and Ausubel, supra. Anyof the identified sequences can be incorporated into a cassette orvector, e.g., for expression in plants. A number of expression vectorssuitable for stable transformation of plant cells or for theestablishment of transgenic plants have been described including thosedescribed in Weissbach and Weissbach, (1989) Methods for Plant MolecularBiology, Academic Press, and Gelvin et al., (1990) Plant MolecularBiology Manual, Kluwer Academic Publishers. Specific examples includethose derived from a Ti plasmid of Agrobacterium tumefaciens, as well asthose disclosed by Herrera-Estrella et al. (1983) Nature 303: 209,Bevan(1984) Nucl Acid Res. 12:8711–8721, Klee(1985) Bio/Technology 3:637–642, for dicotyledonous plants.

Alternatively, non-Ti vectors can be used to transfer the DNA intomonocotyledonous plants and cells by using free DNA delivery techniques.Such methods can involve, for example, the use of liposomes,electroporation, microprojectile bombardment, silicon carbide whiskers,and viruses. By using these methods transgenic plants such as wheat,rice (Christou (1991) Bio/Technology 9: 957–962) and corn (Gordon-Kamm(1990) Plant Cell 2: 603–618) can be produced. An immature embryo canalso be a good target tissue for monocots for direct DNA deliverytechniques by using the particle gun (Weeks et al. (1993) Plant Physiol102: 1077–1084; Vasil (1993) Bio/Technology 10: 667–674; Wan and Lemeaux(1994) Plant Physiol 104: 37–48, and for Agrobacterium-mediated DNAtransfer (Ishida et al. (1996) Nature Biotech 14: 745–750).

Typically, plant transformation vectors include one or more cloned plantcoding sequence (genomic or cDNA) under the transcriptional control of5′ and 3′ regulatory sequences and a dominant selectable marker. Suchplant transformation vectors typically also contain a promoter (e.g., aregulatory region controlling inducible or constitutive,environmentally-or developmentally-regulated, or cell-or tissue-specificexpression), a transcription initiation start site, an RNA processingsignal (such as intron splice sites), a transcription termination site,and/or a polyadenylation signal.

Examples of constitutive plant promoters which can be useful forexpressing the TF sequence include: the cauliflower mosaic virus (CaMV)35S promoter, which confers constitutive, high-level expression in mostplant tissues (see, e.g., Odell et al. (1985) Nature 313:810–812); thenopaline synthase promoter (An et al. (1988) Plant Physiol 88:547–552);and the octopine synthase promoter (Fromm et al. (1989) Plant Cell 1:977–984).

A variety of plant gene promoters that regulate gene expression inresponse to environmental, hormonal, chemical, developmental signals,and in a tissue-active manner can be used for expression of a TFsequence in plants. Choice of a promoter is based largely on thephenotype of interest and is determined by such factors as tissue (e.g.,seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.),inducibility (e.g., in response to wounding, heat, cold, drought, light,pathogens, etc.), timing, developmental stage, and the like. Numerousknown promoters have been characterized and can favorably be employed topromote expression of a polynucleotide of the invention in a transgenicplant or cell of interest. For example, tissue specific promotersinclude: seed-specific promoters (such as the napin, phaseolin or DC3promoter described in U.S. Pat. No. 5,773,697), fruit-specific promotersthat are active during fruit ripening (such as the dru 1 promoter (U.S.Pat. No. 5,783,393), or the 2A 11 promoter (U.S. Pat. No. 4,943,674) andthe tomato polygalacturonase promoter (Bird et al. (1988) Plant Mol Biol11:651), root-specific promoters, such as those disclosed in U.S. Pat.Nos. 5,618,988, 5,837,848 and 5,905,186, pollen-active promoters such asPTA29, PTA26 and PTA13 (U.S. Pat. No. 5,792,929), promoters active invascular tissue (Ringli and Keller (1998) Plant Mol Biol 37:977–988),flower-specific (Kaiser et al, (1995) Plant Mol Biol 28:231–243), pollen(Baerson et al. (1994) Plant Mol Biol 26:1947–1959), carpels (Ohl et al.(1990) Plant Cell 2:837–848), pollen and ovules (Baerson et al. (1993)Plant Mol Biol 22:255–267), auxin-inducible promoters (such as thatdescribed in van der Kop et al. (1999) Plant Mol Biol 39:979–990 orBaumann et al. (1999) Plant Cell 11:323–334), cytokinin-induciblepromoter (Guevara-Garcia (1998) Plant Mol Biol 38:743–753), promotersresponsive to gibberellin (Shi et al. (1998) Plant Mol Biol38:1053–1060, Willmott et al. (1998) 38:817–825) and the like.Additional promoters are those that elicit expression in response toheat (Ainley et al. (1993) Plant Mol Biol 22: 13–23), light (e.g., thepea rbcS-3A promoter, Kuhlemeier et al. (1989) Plant Cell 1:471, and themaize rbcS promoter, Schaffner and Sheen (1991) Plant Cell 3: 997);wounding (e.g., wunI, Siebertz et al. (1989) Plant Cell 1: 961);pathogens (such as the PR-1 promoter described in Buchel et al. (1999)Plant Mol. Biol. 40:387–396, and the PDF1.2 promoter described inManners et al. (1998) Plant Mol. Biol. 38:1071–80), and chemicals suchas methyl jasmonate or salicylic acid (Gatz et al. (1997) Plant Mol Biol48: 89–108). In addition, the timing of the expression can be controlledby using promoters such as those acting at senescence (An and Amazon(1995) Science 270: 1986–1988); or late seed development (Odell et al.(1994) Plant Physiol 106:447–458).

Plant expression vectors can also include RNA processing signals thatcan be positioned within, upstream or downstream of the coding sequence.In addition, the expression vectors can include additional regulatorysequences from the 3′-untranslated region of plant genes, e.g., a 3′terminator region to increase mRNA stability of the mRNA, such as thePI-II terminator region of potato or the octopine or nopaline synthase3′ terminator regions.

Additional Expression Elements

Specific initiation signals can aid in efficient translation of codingsequences. These signals can include, e.g., the ATG initiation codon andadjacent sequences. In cases where a coding sequence, its initiationcodon and upstream sequences are inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only coding sequence (e.g., a matureprotein coding sequence), or a portion thereof, is inserted, exogenoustranscriptional control signals including the ATG initiation codon canbe separately provided. The initiation codon is provided in the correctreading frame to facilitate transcription. Exogenous transcriptionalelements and initiation codons can be of various origins, both naturaland synthetic. The efficiency of expression can be enhanced by theinclusion of enhancers appropriate to the cell system in use.

Expression Hosts

The present invention also relates to host cells which are transducedwith vectors of the invention, and the production of polypeptides of theinvention (including fragments thereof) by recombinant techniques. Hostcells are genetically engineered (i.e., nucleic acids are introduced,e.g., transduced, transformed or transfected) with the vectors of thisinvention, which may be, for example, a cloning vector or an expressionvector comprising the relevant nucleic acids herein. The vector isoptionally a plasmid, a viral particle, a phage, a naked nucleic acid,etc. The engineered host cells can be cultured in conventional nutrientmedia modified as appropriate for activating promoters, selectingtransformants, or amplifying the relevant gene. The culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression, and will be apparent to those skilledin the art and in the references cited herein, including, Sambrook andAusubel.

The host cell can be a eukaryotic cell, such as a yeast cell, or a plantcell, or the host cell can be a prokaryotic cell, such as a bacterialcell. Plant protoplasts are also suitable for some applications. Forexample, the DNA fragments are introduced into plant tissues, culturedplant cells or plant protoplasts by standard methods includingelectroporation (Fromm et al., (1985) Proc. Natl. Acad. Sci. USA 82,5824, infection by viral vectors such as cauliflower mosaic virus (CaMV)(Hohn et al., (1982) Molecular Biology of Plant Tumors, (Academic Press,New York) pp. 549–560; U.S. Pat. No. 4,407,956), high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,(1987) Nature 327, 70–73), use of pollen as vector (WO 85/01856), or useof Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmidin which DNA fragments are cloned. The T-DNA plasmid is transmitted toplant cells upon infection by Agrobacterium tumefaciens, and a portionis stably integrated into the plant genome (Horsch et al. (1984) Science233:496–498; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80, 4803).

The cell can include a nucleic acid of the invention which encodes apolypeptide, wherein the cell expresses a polypeptide of the invention.The cell can also include vector sequences, or the like. Furthermore,cells and transgenic plants that include any polypeptide or nucleic acidabove or throughout this specification, e.g., produced by transductionof a vector of the invention, are an additional feature of theinvention.

For long-term, high-yield production of recombinant proteins, stableexpression can be used. Host cells transformed with a nucleotidesequence encoding a polypeptide of the invention are optionally culturedunder conditions suitable for the expression and recovery of the encodedprotein from cell culture. The protein or fragment thereof produced by arecombinant cell may be secreted, membrane-bound, or containedintracellularly, depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining polynucleotides encoding mature proteins of the invention canbe designed with signal sequences which direct secretion of the maturepolypeptides through a prokaryotic or eukaryotic cell membrane.

Modified Amino Acid Residues

Polypeptides of the invention may contain one or more modified aminoacid residues. The presence of modified amino acids may be advantageousin, for example, increasing polypeptide half-life, reducing polypeptideantigenicity or toxicity, increasing polypeptide storage stability, orthe like. Amino acid residue(s) are modified, for example,co-translationally or post-translationally during recombinant productionor modified by synthetic or chemical means.

Non-limiting examples of a modified amino acid residue includeincorporation or other use of acetylated amino acids, glycosylated aminoacids, sulfated amino acids, prenylated (e.g., farnesylated,geranylgeranylated) amino acids, PEG modified (e.g., “PEGylated”) aminoacids, biotinylated amino acids, carboxylated amino acids,phosphorylated amino acids, etc. References adequate to guide one ofskill in the modification of amino acid residues are replete throughoutthe literature.

The modified amino acid residues may prevent or increase affinity of thepolypeptide for another molecule, including, but not limited to,polynucleotide, proteins, carbohydrates, lipids and lipid derivatives,and other organic or synthetic compounds.

Identification of Additional Factors

A transcription factor provided by the present invention can also beused to identify additional endogenous or exogenous molecules that canaffect a phentoype or trait of interest. On the one hand, such moleculesinclude organic (small or large molecules) and/or inorganic compoundsthat affect expression of (i.e., regulate) a particular transcriptionfactor. Alternatively, such molecules include endogenous molecules thatare acted upon either at a transcriptional level by a transcriptionfactor of the invention to modify a phenotype as desired. For example,the transcription factors can be employed to identify one or moredownstream gene with which is subject to a regulatory effect of thetranscription factor. In one approach, a transcription factor ortranscription factor homologue of the invention is expressed in a hostcell, e.g., a transgenic plant cell, tissue or explant, and expressionproducts, either RNA or protein, of likely or random targets aremonitored, e.g., by hybridization to a microarray of nucleic acid probescorresponding to genes expressed in a tissue or cell type of interest,by two-dimensional gel electrophoresis of protein products, or by anyother method known in the art for assessing expression of gene productsat the level of RNA or protein. Alternatively, a transcription factor ofthe invention can be used to identify promoter sequences (i.e., bindingsites) involved in the regulation of a downstream target. Afteridentifying a promoter sequence, interactions between the transcriptionfactor and the promoter sequence can be modified by changing specificnucleotides in the promoter sequence or specific amino acids in thetranscription factor that interact with the promoter sequence to alter aplant trait. Typically, transcription factor DNA-binding sites areidentified by gel shift assays. After identifying the promoter regions,the promoter region sequences can be employed in double-stranded DNAarrays to identify molecules that affect the interactions of thetranscription factors with their promoters (Bulyk et al. (1999) NatureBiotechnology 17:573–577).

The identified transcription factors are also useful to identifyproteins that modify the activity of the transcription factor. Suchmodification can occur by covalent modification, such as byphosphorylation, or by protein-protein (homo or heteropolymer)interactions. Any method suitable for detecting protein-proteininteractions can be employed. Among the methods that can be employed areco-immunoprecipitation, cross-linking and co-purification throughgradients or chromatographic columns, and the two-hybrid yeast system.

The two-hybrid system detects protein interactions in vivo and isdescribed in Chien et al. ((1991), Proc. Natl. Acad. Sci. USA88:9578–9582) and is commercially available from Clontech (Palo Alto,Calif.). In such a system, plasmids are constructed that encode twohybrid proteins: one consists of the DNA-binding domain of atranscription activator protein fused to the TF polypeptide and theother consists of the transcription activator protein's activationdomain fused to an unknown protein that is encoded by a cDNA that hasbeen recombined into the plasmid as part of a cDNA library. TheDNA-binding domain fusion plasmid and the cDNA library are transformedinto a strain of the yeast Saccharomyces cerevisiae that contains areporter gene (e.g., lacZ) whose regulatory region contains thetranscription activator's binding site. Either hybrid protein alonecannot activate transcription of the reporter gene. Interaction of thetwo hybrid proteins reconstitutes the functional activator protein andresults in expression of the reporter gene, which is detected by anassay for the reporter gene product. Then, the library plasmidsresponsible for reporter gene expression are isolated and sequenced toidentify the proteins encoded by the library plasmids. After identifyingproteins that interact with the transcription factors, assays forcompounds that interfere with the TF protein-protein interactions can bepreformed.

Identification of Modulators

In addition to the intracellular molecules described above,extracellular molecules that alter activity or expression of atranscription factor, either directly or indirectly, can be identified.For example, the methods can entail first placing a candidate moleculein contact with a plant or plant cell. The molecule can be introduced bytopical administration, such as spraying or soaking of a plant, and thenthe molecule's effect on the expression or activity of the TFpolypeptide or the expression of the polynucleotide monitored. Changesin the expression of the TF polypeptide can be monitored by use ofpolyclonal or monoclonal antibodies, gel electrophoresis or the like.Changes in the expression of the corresponding polynucleotide sequencecan be detected by use of microarrays, Northerns, quantitative PCR, orany other technique for monitoring changes in mRNA expression. Thesetechniques are exemplified in Ausubel et al. (eds) Current Protocols inMolecular Biology, John Wiley & Sons (1998, and supplements through2001). Such changes in the expression levels can be correlated withmodified plant traits and thus identified molecules can be useful forsoaking or spraying on fruit, vegetable and grain crops to modify traitsin plants.

Essentially any available composition can be tested for modulatoryactivity of expression or activity of any nucleic acid or polypeptideherein. Thus, available libraries of compounds such as chemicals,polypeptides, nucleic acids and the like can be tested for modulatoryactivity. Often, potential modulator compounds can be dissolved inaqueous or organic (e.g., DMSO-based) solutions for easy delivery to thecell or plant of interest in which the activity of the modulator is tobe tested. Optionally, the assays are designed to screen large modulatorcomposition libraries by automating the assay steps and providingcompounds from any convenient source to assays, which are typically runin parallel (e.g., in microtiter formats on microtiter plates in roboticassays).

In one embodiment, high throughput screening methods involve providing acombinatorial library containing a large number of potential compounds(potential modulator compounds). Such “combinatorial chemical libraries”are then screened in one or more assays, as described herein, toidentify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as target compounds.

A combinatorial chemical library can be, e.g., a collection of diversechemical compounds generated by chemical synthesis or biologicalsynthesis. For example, a combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (e.g., in one example, amino acids) in every possible way for agiven compound length (i.e., the number of amino acids in a polypeptidecompound of a set length). Exemplary libraries include peptidelibraries, nucleic acid libraries, antibody libraries (see, e.g., Vaughnet al. (1996) Nature Biotechnology, 14(3):309–314 and PCT/US96/10287),carbohydrate libraries (see, e.g., Liang et al. Science (1996)274:1520–1522 and U.S. Pat. No. 5,593,853), peptide nucleic acidlibraries (see, e.g., U.S. Pat. No. 5,539,083), and small organicmolecule libraries (see, e.g., benzodiazepines, Baum C&EN January 18,page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinonesand metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat.Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. 5,506,337)and the like.

Preparation and screening of combinatorial or other libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175; Furka, (1991) Int. J. Pept. Prot. Res.37:487–493; and Houghton et al. (1991) Nature 354:84–88). Otherchemistries for generating chemical diversity libraries can also beused.

In addition, as noted, compound screening equipment for high-throughputscreening is generally available, e.g., using any of a number of wellknown robotic systems that have also been developed for solution phasechemistries useful in assay systems. These systems include automatedworkstations including an automated synthesis apparatus and roboticsystems utilizing robotic arms. Any of the above devices are suitablefor use with the present invention, e.g., for high-throughput screeningof potential modulators. The nature and implementation of modificationsto these devices (if any) so that they can operate as discussed hereinwill be apparent to persons skilled in the relevant art.

Indeed, entire high throughput screening systems are commerciallyavailable. These systems typically automate entire procedures includingall sample and reagent pipetting, liquid dispensing, timed incubations,and final readings of the microplate in detector(s) appropriate for theassay. These configurable systems provide high throughput and rapidstart up as well as a high degree of flexibility and customization.Similarly, microfluidic implementations of screening are alsocommercially available.

The manufacturers of such systems provide detailed protocols the varioushigh throughput. Thus, for example, Zymark Corp. provides technicalbulletins describing screening systems for detecting the modulation ofgene transcription, ligand binding, and the like. The integrated systemsherein, in addition to providing for sequence alignment and, optionally,synthesis of relevant nucleic acids, can include such screeningapparatus to identify modulators that have an effect on one or morepolynucleotides or polypeptides according to the present invention.

In some assays it is desirable to have positive controls to ensure thatthe components of the assays are working properly. At least two types ofpositive controls are appropriate. That is, known transcriptionalactivators or inhibitors can be incubated with cells/plants/etc. in onesample of the assay, and the resulting increase/decrease intranscription can be detected by measuring the resulting increase inRNA/protein expression, etc., according to the methods herein. It willbe appreciated that modulators can also be combined with transcriptionalactivators or inhibitors to find modulators that inhibit transcriptionalactivation or transcriptional repression. Either expression of thenucleic acids and proteins herein or any additional nucleic acids orproteins activated by the nucleic acids or proteins herein, or both, canbe monitored.

In an embodiment, the invention provides a method for identifyingcompositions that modulate the activity or expression of apolynucleotide or polypeptide of the invention. For example, a testcompound, whether a small or large molecule, is placed in contact with acell, plant (or plant tissue or explant), or composition comprising thepolynucleotide or polypeptide of interest and a resulting effect on thecell, plant, (or tissue or explant) or composition is evaluated bymonitoring, either directly or indirectly, one or more of: expressionlevel of the polynucleotide or polypeptide, activity (or modulation ofthe activity) of the polynucleotide or polypeptide. In some cases, analteration in a plant phenotype can be detected following contact of aplant (or plant cell, or tissue or explant) with the putative modulator,e.g., by modulation of expression or activity of a polynucleotide orpolypeptide of the invention. Modulation of expression or activity of apolynucleotide or polypeptide of the invention may also be caused bymolecular elements in a signal transduction second messenger pathway andsuch modulation can affect similar elements in the same or anothersignal transduction second messenger pathway.

Subsequences

Also contemplated are uses of polynucleotides, also referred to hereinas oligonucleotides, typically having at least 12 bases, preferably atleast 15, more preferably at least 20, 30, or 50 bases, which hybridizeunder at least highly stringent (or ultra-high stringent orultra-ultra-high stringent conditions) conditions to a polynucleotidesequence described above. The polynucleotides may be used as probes,primers, sense and antisense agents, and the like, according to methodsas noted supra.

Subsequences of the polynucleotides of the invention, includingpolynucleotide fragments and oligonucleotides are useful as nucleic acidprobes and primers. An oligonucleotide suitable for use as a probe orprimer is at least about 15 nucleotides in length, more often at leastabout 18 nucleotides, often at least about 21 nucleotides, frequently atleast about 30 nucleotides, or about 40 nucleotides, or more in length.A nucleic acid probe is useful in hybridization protocols, e.g., toidentify additional polypeptide homologues of the invention, includingprotocols for microarray experiments. Primers can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, e.g., by thepolymerase chain reaction (PCR) or other nucleic-acid amplificationmethods. See Sambrook and Ausubel, supra.

In addition, the invention includes an isolated or recombinantpolypeptide including a subsequence of at least about 15 contiguousamino acids encoded by the recombinant or isolated polynucleotides ofthe invention. For example, such polypeptides, or domains or fragmentsthereof, can be used as immunogens, e.g., to produce antibodies specificfor the polypeptide sequence, or as probes for detecting a sequence ofinterest. A subsequence can range in size from about 15 amino acids inlength up to and including the full length of the polypeptide.

To be encompassed by the present invention, an expressed polypeptidewhich comprises such a polypeptide subsequence performs at least onebiological function of the intact polypeptide in substantially the samemanner, or to a similar extent, as does the intact polypeptide. Forexample, a polypeptide fragment can comprise a recognizable structuralmotif or functional domain such as a DNA binding domain that binds to aspecific DNA promoter region, an activation domain or a domain forprotein-protein interactions.

Production of Transgenic Plants

Modification of Traits

The polynucleotides of the invention are favorably employed to producetransgenic plants with various traits, or characteristics, that havebeen modified in a desirable manner, e.g., to improve the seedcharacteristics of a plant. For example, alteration of expression levelsor patterns (e.g., spatial or temporal expression patterns) of one ormore of the transcription factors (or transcription factor homologues)of the invention, as compared with the levels of the same protein foundin a wild type plant, can be used to modify a plant's traits. Anillustrative example of trait modification, improved characteristics, byaltering expression levels of a particular transcription factor isdescribed further in the Examples and the Sequence Listing.

Arabidopsis as a Model System

Arabidopsis thaliana is the object of rapidly growing attention as amodel for genetics and metabolism in plants. Arabidopsis has a smallgenome, and well documented studies are available. It is easy to grow inlarge numbers and mutants defining important genetically controlledmechanisms are either available, or can readily be obtained. Variousmethods to introduce and express isolated homologous genes are available(see Koncz, et al., eds. Methods in Arabidopsis Research. et al. (1992),World Scientific, New Jersey, N.J., in “Preface”). Because of its smallsize, short life cycle, obligate autogamy and high fertility,Arabidopsis is also a choice organism for the isolation of mutants andstudies in morphogenetic and development pathways, and control of thesepathways by transcription factors (Koncz, supra, p. 72). A number ofstudies introducing transcription factors into A. thaliana havedemonstrated the utility of this plant for understanding the mechanismsof gene regulation and trait alteration in plants. See, for example,Koncz, supra, and U.S. Pat. No. 6,417,428).

Arabidopsis Genes in Transgenic Plants.

Expression of genes which encode transcription factors modify expressionof endogenous genes, polynucleotides, and proteins are well known in theart. In addition, transgenic plants comprising isolated polynucleotidesencoding transcription factors may also modify expression of endogenousgenes, polynucleotides, and proteins. Examples include Peng et al.(1997, Genes and Development 11:3194–3205) and Peng et al. (1999,Nature, 400:256–261). In addition, many others have demonstrated that anArabidopsis transcription factor expressed in an exogenous plant specieselicits the same or very similar phenotypic response. See, for example,Fu et al. (2001, Plant Cell 13:1791–1802); Nandi et al. (2000, Curr.Biol. 10:215–218); Coupland (1995, Nature 377:482–483); and Weigel andNilsson (1995, Nature 377:482–500).

Homologous Genes Introduced into Transgenic Plants.

Homologous genes that may be derived from any plant, or from any sourcewhether natural, synthetic, semi-synthetic or recombinant, and thatshare significant sequence identity or similarity to those provided bythe present invention, may be introduced into plants, for example, cropplants, to confer desirable or improved traits. Consequently, transgenicplants may be produced that comprise a recombinant expression vector orcassette with a promoter operably linked to one or more sequenceshomologous to presently disclosed sequences. The promoter may be, forexample, a plant or viral promoter.

The invention thus provides for methods for preparing transgenic plants,and for modifying plant traits. These methods include introducing into aplant a recombinant expression vector or cassette comprising afunctional promoter operably linked to one or more sequences homologousto presently disclosed sequences. Plants and kits for producing theseplants that result from the application of these methods are alsoencompassed by the present invention.

Traits of Interest

Examples of some of the traits that may be desirable in plants, and thatmay be provided by transforming the plants with the presently disclosedsequences, are listed in Table 6.

TABLE 6 Trait Transcription factor genes Utility Gene Category Traitsthat impact traits effect on: Resistance and Salt stress G22; G196;G226; G303; G312; Germination rate, tolerance resistance G325; G353;G482; G545; G801; survivability, yield; G867; G884; G922; G926; G1452;extended growth G1794; G1820; G1836; G1843; range G1863; G2053; G2110;G2140; G2153; G2379; G2701; G2713; G2719; G2789 Osmotic stress G47;G175; G188; G303; G325; Germination rate, resistance G353; G489; G502;G526; G921; survivability, yield G922; G926; G1069; G1089; G1452; G1794;G1930; G2140; G2153; G2379; G2701; G2719; G2789; Cold stress G256; G394;G664; G867; G1322; Germination, resistance; G2130 growth, earlier coldgermination planting Tolerance to freezing G303; G325; G353; G720; G912;Survivability, yield, G913; G1794; G2053; G2140; appearance, G2153;G2379; G2701; G2719; extended range G2789 Heat stress resistance G3;G464; G682; G864; G964; Germination, G1305; G1645; G2130 G2430 growth,later planting Drought, low humidity G303; G325; G353; G720; G912;Survivability, yield, resistance G926; G1452; G1794; G1820; extendedrange G1843; G2053; G2140; G2153; G2379; G2583; G2701; G2719; G2789Radiation resistance G1052 Survivability, vigor, appearance Decreasedherbicide G343; G2133; G2517 Resistant to sensitivity increasedherbicide use Increased herbicide G374; G877; G1519 Use as a herbicidesensitivity target Oxidative stress G477; G789; G1807; G2133; Improvedyield, G2517 appearance, reduced senescence Light response G183; G354;G375; G1062; Germination, G1322; G1331; G1488; G1494; growth, G1521;G1786; G1794; G2144; development, G2555; flowering time Development,Overall plant G24; G27; G31; G33; G47; G147; Vascular tissues,morphology architecture G156; G160; G182; G187; G195; lignin content;cell G196; G211; G221; G237; G280; wall content; G342; G352; G357; G358;G360; appearance G362; G364; G365; G367; G373; G377; G396; G431; G447;G479; G546; G546; G551; G578; G580; G596; G615; G617; G620; G625; G638;G658; G716; G725; G727; G730; G740; G770; G858; G865; G869; G872; G904;G910; G912; G920; G939; G963; G977; G979; G987; G988; G993; G1007;G1010; G1014; G1035; G1046; G1049; G1062; G1069; G1070; G1076; G1089;G1093; G1127; G1131; G1145; G1229; G1246; G1304; G1318; G1320; G1330;G1331; G1352; G1354; G1360; G1364; G1379; G1384; G1399; G1415; G1417;G1442; G1453; G1454; G1459; G1460; G1471; G1475; G1477; G1487; G1487;G1492; G1499; G1499; G1531; G1540; G1543; G1543; G1544; G1548; G1584;G1587; G1588; G1589; G1636; G1642; G1747; G1749; G1749; G1751; G1752;G1763; G1766; G1767; G1778; G1789; G1790; G1791; G1793; G1794; G1795;G1800; G1806; G1811; G1835; G1836; G1838; G1839; G1843; G1853; G1855;G1865; G1881; G1882; G1883; G1884; G1891; G1896; G1898; G1902; G1904;G1906; G1913; G1914; G1925; G1929; G1930; G1954; G1958; G1965; G1976;G2057; G2107; G2133; G2134; G2151; G2154; G2157; G2181; G2290; G2299;G2340; G2340; G2346; G2373; G2376; G2424; G2465; G2505; G2509; G2512;G2513; G2519; G2520; G2533; G2534; G2573; G2589; G2687; G2720; G2787;G2789; G2893 Size: increased stature G189; G1073; G1435; G2430 Size;reduced stature or G3; G5; G21; G23; G39; G165; Ornamental; smalldwarfism G184; G194; G258; G280; G340; stature provides G343; G353;G354; G362; G363; wind resistance; G370; G385; G396; G439; G440;creation of dwarf G447; G450; G550; G557; G599; varieties G636; G652;G670; G671; G674; G729; G760; G804; G831; G864; G884; G898; G900; G912;G913; G922; G932; G937; G939; G960; G962; G977; G991; G1000; G1008;G1020; G1023; G1053; G1067; G1075; G1137; G1181; G1198; G1228; G1266;G1267; G1275; G1277; G1309; G1311; G1314; G1317; G1322; G1323; G1326;G1332; G1334; G1367; G1381; G1382; G1386; G1421; G1488; G1494; G1537;G1545; G1560; G1586; G1641; G1652; G1655; G1671; G1750; G1756; G1757;G1782; G1786; G1794; G1839; G1845; G1879; G1886; G1888; G1933; G1939;G1943; G1944; G2011; G2094; G2115; G2130; G2132; G2144; G2145; G2147;G2156; G2294; G2313; G2344; G2431; G2510; G2517; G2521; G2893; G2893Fruit size and number G362 Biomass, yield, cotton boll fiber densityFlower structure, G47; G259; G353; G354; G671; Ornamental inflorescenceG732; G988; G1000; G1063; horticulture; G1140; G1326; G1449; G1543;production of G1560; G1587; G1645; G1947; saffron or other G2108; G2143;G2893 edible flowers Number and G225; G226; G247; G362; G585; Resistanceto pests development of G634; G676; G682; G1014; and desiccation;trichomes G1332; G1452; G1795; G2105 essential oil production Seed size,color, and G156; G450; G584; G652; G668; Yield number G858; G979; G1040;G1062; G1145; G1255; G1494; G1531; G1534; G1594; G2105; G2114; Rootdevelopment, G9; G1482; G1534; G1794; modifications G1852; G2053; G2136;G2140 Modifications to root G225; G226 Nutrient, water hairs uptake,pathogen resistance Apical dominance G559; G732; G1255; G1275;Ornamental G1411; G1488; G1635; G2452; horticulture G2509 Branchingpatterns G568; G988; G1548 Ornamental horticulture, knot reduction,improved windscreen Leaf shape, color, G375; G377; G428; G438; G447;Appealing shape or modifications G464; G557; G577; G599; G635 shinyleaves for G671; G674; G736; G804; G903; ornamental G977; G921; G922;G1038; agriculture, G1063; G1067; G1073; G1075; increased biomass G1146;G1152; G1198; G1267; or photosynthesis G1269; G1452; G1484; G1586;G1594; G1767; G1786; G1792; G1886; G2059; G2094; G2105; G2113; G2117;G2143; G2144; G2431; G2452; G2465; G2587; G2583; G2724; Silique G1134Ornamental Stem morphology G47; G438; G671; G748; G988; Ornamental;G1000 digestibility Shoot modifications G390; G391 Ornamental stembifurcations Disease, Pathogen Bacterial G211; G347; G367; G418; G525;Yield, appearance, Resistance G545; G578; G1049 survivability, extendedrange Fungal G19; G28; G28; G28; G147; Yield, appearance, G188; G207;G211; G237; G248; survivability, G278; G347; G367; G371; G378; extendedrange G409; G477; G545; G545; G558; G569; G578; G591; G594; G616; G789;G805; G812; G865; G869; G872; G881; G896; G940; G1047; G1049; G1064;G1084; G1196; G1255; G1266; G1363; G1514; G1756; G1792; G1792; G1792;G1792; G1880; G1919; G1919; G1927; G1927; G1936; G1936; G1950; G2069;G2130; G2380; G2380; G2555 Nutrients Increased tolerance to G225; G226;G1792 nitrogen-limited soils Increased tolerance to G419; G545; G561;G1946 phosphate-limited soils Increased tolerance to G561; G911potassium-limited soils Hormonal Hormone sensitivity G12; G546; G926;G760; G913; Seed dormancy, G926; G1062; G1069; G1095; drought tolerance;G1134; G1330; G1452; G1666; plant form, fruit G1820; G2140; G2789ripening Seed biochemistry Production of seed G214; G259; G490; G652;G748; Antioxidant activity, prenyl lipids, including G883; G1052; G1328;G1930; vitamin E tocopherol G2509; G2520 Production of seed G20Precursors for sterols human steroid hormones; cholesterol modulatorsProduction of seed G353; G484; G674; G1272; Defense againstglucosinolates G1506; G1897; G1946; G2113; insects; putative G2117;G2155; G2290; G2340 anticancer activity; undesirable in animal feedsModified seed oil G162; G162; G180; G192; G241; Vegetable oil contentG265; G286; G291; G427; G509; production; G519; G561; G567; G590; G818;increased caloric G849; G892; G961; G974; G1063; value for animal G1143;G1190; G1198; G1226; feeds; lutein content G1229; G1323; G1451; G1471;G1478; G1496; G1526; G1543; G1640; G1644; G1646; G1672; G1677; G1750;G1765; G1777; G1793; G1838; G1902; G1946; G1948; G2059; G2123; G2138;G2139; G2343; G2792; G2830 Modified seed oil G217; G504; G622; G778;G791; Heat stability, composition G861; G869; G938; G965; G1417;digestibility of seed G2192 oils Modified seed protein G162; G226; G241;G371; G427; Reduced caloric content G509; G567; G597; G732; G849; valuefor humans G865; G892; G963; G988; G1323; G1323; G1419; G1478; G1488;G1634; G1637; G1641; G1644; G1652; G1677; G1777; G1777; G1818; G1820;G1903; G1909; G1946; G1946; G1958; G2059; G2117; G2417; G2509 Leafbiochemistry Production of flavonoids G1666* Ornamental pigmentproduction; pathogen resistance; health benefits Production of leafG264; G353; G484; G652; G674; Defense against glucosinolates G681;G1069; G1198; G1322; insects; putative G1421; G1657; G1794; G1897;anticancer activity; G1946; G2115; G2117; G2144; undesirable in G2155;G2155; G2340; G2512; animal feeds G2520; G2552 Production of diterpenesG229 Induction of enzymes involved in alkaloid biosynthesis Productionof G546 Ornamental pigment anthocyanin Production of leaf G561; G2131;G2424 Precursors for phytosterols, inc. human steroid stigmastanol,hormones; campestrol cholesterol modulators Leaf fatty acid G214; G377;G861; G962; G975; Nutritional value; composition G987; G1266; G1337;G1399; increase in waxes G1465; G1512; G2136; G2147; for disease G2192resistance Production of leaf G214; G259; G280; G652; G987; Antioxidantactivity, prenyl lipids, including G1543; G2509; G2520 vitamin Etocopherol Biochemistry, Production of G229; G663 general miscellaneoussecondary metabolites Sugar, starch, G158; G211; G211; G237; G242; Fooddigestibility, hemicellulose G274; G598; G1012; G1266; hemicellulose &composition, G1309; G1309; G1641; G1765; pectin content; fiber G1865;G2094; G2094; G2589; content; plant G2589 tensile strength, woodquality, pathogen resistance, pulp production; tuber starch contentSugar sensing Plant response to sugars G26; G38; G43; G207; G218;Photosynthetic rate, G241; G254; G263; G308; G536; carbohydrate G567;G567; G680; G867; G912; accumulation, G956; G996; G1068; G1225; biomassproduction, G1314; G1314; G1337; G1759; source-sink G1804; G2153; G2379relationships, senescence Growth, Plant growth rate and G447; G617;G674; G730; G917; Faster growth, Reproduction development G937; G1035;G1046; G1131; increased biomass G1425; G1452; G1459; G1492; or yield,improved G1589; G1652; G1879; G1943; appearance; delay in G2430; G2431;G2465; G2521 bolting Embryo development G167 Seed germination rate G979;G1792; G2130 Yield Plant, seedling vigor G561; G2346 Survivability,yield Senescence; cell death G571; G636; G878; G1050; Yield, appearance;G1463; G1749; G1944; G2130; response to G2155; G2340; G2383 pathogens;Modified fertility G39; G340; G439; G470; G559; Prevents or G615; G652;G671; G779; G962; minimizes escape of G977; G988; G1000; G1063; thepollen of GMOs G1067; G1075; G1266; G1311; G1321; G1326; G1367; G1386;G1421; G1453; G1471; G1453; G1560; G1594; G1635; G1750; G1947; G2011;G2094; G2113; G2115; G2130; G2143; G2147; G2294; G2510; G2893 Earlyflowering G147; G157; G180; G183; G183; Faster generation G184; G185;G208; G227; G294; time; synchrony of G390; G390; G390; G391; G391;flowering; potential G427; G427; G490; G565; G590; for introducing newG592; G720; G789; G865; G898; traits to single G898; G989; G989; G1037;variety G1037; G1142; G1225; G1225; G1226; G1242; G1305; G1305; G1380;G1380; G1480; G1480; G1488; G1494; G1545; G1545; G1649; G1706; G1760;G1767; G1767; G1820; G1841; G1841; G1842; G1843; G1843; G1946; G1946;G2010; G2030; G2030; G2144; G2144; G2295; G2295; G2347; G2348; G2348;G2373; G2373; G2509; G2509; G2555; G2555 Delayed flowering G8; G47;G192; G214; G234; Delayed time to G361; G362; G562; G568; G571; pollenproduction of G591; G680; G736; G748; G859; GMO plants; G878; G910;G912; G913; G971; synchrony of G994; G1051; G1052; G1073; flowering;increased G1079; G1335; G1435; G1452; yield G1478; G1789; G1804; G1865;G1865; G1895; G1900; G2007; G2133; G2155; G2291; G2465 Extendedflowering G1947 phase Flower and leaf G259; G353; G377; G580; G638Ornamental development G652; G858; G869; G917; G922; applications; G932;G1063; G1075; G1140; decreased fertility G1425; G1452; G1499; G1548;G1645; G1865; G1897; G1933; G2094; G2124; G2140; G2143; G2535; G2557Flower abscission G1897 Ornamental: longer retention of flowers Whenco-expressed with G669 and G663

When co-expressed with G669 and G663

Significance of Modified Plant Traits

Currently, the existence of a series of maturity groups for differentlatitudes represents a major barrier to the introduction of new valuabletraits. Any trait (e.g. disease resistance) has to be bred into each ofthe different maturity groups separately, a laborious and costlyexercise. The availability of single strain, which could be grown at anylatitude, would therefore greatly increase the potential for introducingnew traits to crop species such as soybean and cotton.

For many of the traits, listed in Table 6 and below, that may beconferred to plants, a single transcription factor gene may be used toincrease or decrease, advance or delay, or improve or prove deleteriousto a given trait. For example, overexpression of a transcription factorgene that naturally occurs in a plant may cause early flowering relativeto non-transformed or wild-type plants. By knocking out the gene, orsuppressing the gene (with, for example, antisense suppression) theplant may experience delayed flowering. Similarly, overexpressing orsuppressing one or more genes can impart significant differences inproduction of plant products, such as different fatty acid ratios. Thus,suppressing a gene that causes a plant to be more sensitive to cold mayimprove a plant's tolerance of cold.

Salt stress resistance. Soil salinity is one of the more importantvariables that determines where a plant may thrive. Salinity isespecially important for the successful cultivation of crop plants,particular in many parts of the world that have naturally high soil saltconcentrations, or where the soil has been over-utilized. Thus,presently disclosed transcription factor genes that provide increasedsalt tolerance during germination, the seedling stage, and throughout aplant's life cycle would find particular value for impartingsurvivability and yield in areas where a particular crop would notnormally prosper.

Osmotic stress resistance. Presently disclosed transcription factorgenes that confer resistance to osmotic stress may increase germinationrate under adverse conditions, which could impact survivability andyield of seeds and plants.

Cold stress resistance. The potential utility of presently disclosedtranscription factor genes that increase tolerance to cold is to conferbetter germination and growth in cold conditions. The germination ofmany crops is very sensitive to cold temperatures. Genes that wouldallow germination and seedling vigor in the cold would have highlysignificant utility in allowing seeds to be planted earlier in theseason with a high rate of survivability. Transcription factor genesthat confer better survivability in cooler climates allow a grower tomove up planting time in the spring and extend the growing seasonfurther into autumn for higher crop yields.

Tolerance to freezing. The presently disclosed transcription factorgenes that impart tolerance to freezing conditions are useful forenhancing the survivability and appearance of plants conditions orconditions that would otherwise cause extensive cellular damage. Thus,germination of seeds and survival may take place at temperaturessignificantly below that of the mean temperature required forgermination of seeds and survival of non-transformed plants. As withsalt tolerance, this has the added benefit of increasing the potentialrange of a crop plant into regions in which it would otherwise succumb.Cold tolerant transformed plants may also be planted earlier in thespring or later in autumn, with greater success than withnon-transformed plants.

Heat stress tolerance. The germination of many crops is also sensitiveto high temperatures. Presently disclosed transcription factor genesthat provide increased heat tolerance are generally useful in producingplants that germinate and grow in hot conditions, may find particularuse for crops that are planted late in the season, or extend the rangeof a plant by allowing growth in relatively hot climates.

Drought, low humidity tolerance. Strategies that allow plants to survivein low water conditions may include, for example, reduced surface areaor surface oil or wax production. A number of presently disclosedtranscription factor genes increase a plant's tolerance to low waterconditions and provide the benefits of improved survivability, increasedyield and an extended geographic and temporal planting range.

Radiation resistance. Presently disclosed transcription factor geneshave been shown to increase lutein production. Lutein, like otherxanthophylls such as zeaxanthin and violaxanthin, are important in theprotection of plants against the damaging effects of excessive light.Lutein contributes, directly or indirectly, to the rapid rise ofnon-photochemical quenching in plants exposed to high light. Increasedtolerance of field plants to visible and ultraviolet light impactssurvivability and vigor, particularly for recent transplants. Alsoaffected are the yield and appearance of harvested plants or plantparts. Crop plants engineered with presently disclosed transcriptionfactor genes that cause the plant to produce higher levels of luteintherefore would have improved photoprotection, leading to less oxidativedamage and increase vigor, survivability and higher yields under highlight and ultraviolet light conditions.

Decreased herbicide sensitivity. Presently disclosed transcriptionfactor genes that confer resistance or tolerance to herbicides (e.g.,glyphosate) may find use in providing means to increase herbicideapplications without detriment to desirable plants. This would allow forthe increased use of a particular herbicide in a local environment, withthe effect of increased detriment to undesirable species and less harmto transgenic, desirable cultivars.

Increased herbicide sensitivity. Knockouts of a number of the presentlydisclosed transcription factor genes have been shown to be lethal todeveloping embryos. Thus, these genes are potentially useful asherbicide targets.

Oxidative stress. In plants, as in all living things, abiotic and bioticstresses induce the formation of oxygen radicals, including superoxideand peroxide radicals. This has the effect of accelerating senescence,particularly in leaves, with the resulting loss of yield and adverseeffect on appearance. Generally, plants that have the highest level ofdefense mechanisms, such as, for example, polyunsaturated moieties ofmembrane lipids, are most likely to thrive under conditions thatintroduce oxidative stress (e.g., high light, ozone, water deficit,particularly in combination). Introduction of the presently disclosedtranscription factor genes that increase the level of oxidative stressdefense mechanisms would provide beneficial effects on the yield andappearance of plants. One specific oxidizing agent, ozone, has beenshown to cause significant foliar injury, which impacts yield andappearance of crop and ornamental plants. In addition to reduced foliarinjury that would be found in ozone resistant plant created bytransforming plants with some of the presently disclosed transcriptionfactor genes, the latter have also been shown to have increasedchlorophyll fluorescence (Yu-Sen Chang et al. Bot. Bull. Acad. Sin.(2001) 42: 265–272).

Heavy metal tolerance. Heavy metals such as lead, mercury, arsenic,chromium and others may have a significant adverse impact on plantrespiration. Plants that have been transformed with presently disclosedtranscription factor genes that confer improved resistance to heavymetals, through for example, sequestering or reduced uptake of themetals will show improved vigor and yield in soils with relatively highconcentrations of these elements. Conversely, transgenic transcriptionfactors may also be introduced into plants to confer an increase inheavy metal uptake, which may benefit efforts to clean up contaminatedsoils.

Light response. Presently disclosed transcription factor genes thatmodify a plant's response to light may be useful for modifying a plant'sgrowth or development, for example, photomorphogenesis in poor light, oraccelerating flowering time in response to various light intensities,quality or duration to which a non-transformed plant would not similarlyrespond. Examples of such responses that have been demonstrated includeleaf number and arrangement, and early flower bud appearances.

Overall plant architecture. Several presently disclosed transcriptionfactor genes have been introduced into plants to alter numerous aspectsof the plant's morphology. For example, it has been demonstrated that anumber of transcription factors may be used to manipulate branching,such as the means to modify lateral branching, a possible application inthe forestry industry. Transgenic plants have also been produced thathave altered cell wall content, lignin production, flower organ number,or overall shape of the plants. Presently disclosed transcription factorgenes transformed into plants may be used to affect plant morphology byincreasing or decreasing internode distance, both of which may beadvantageous under different circumstances. For example, for fast growthof woody plants to provide more biomass, or fewer knots, increasedinternode distances are generally desirable. For improved wind screeningof shrubs or trees, or harvesting characteristics of, for example,members of the Gramineae family, decreased internode distance may beadvantageous. These modifications would also prove useful in theornamental horticulture industry for the creation of unique phenotypiccharacteristics of ornamental plants.

Increased stature. For some ornamental plants, the ability to providelarger varieties may be highly desirable. For many plants, including tfruit-bearing trees or trees and shrubs that serve as view or windscreens, increased stature provides obvious benefits. Crop species mayalso produce higher yields on larger cultivars.

Reduced stature or dwarfism. Presently disclosed transcription factorgenes that decrease plant stature can be used to produce plants that aremore resistant to damage by wind and rain, or more resistant to heat orlow humidity or water deficit. Dwarf plants are also of significantinterest to the ornamental horticulture industry, and particularly forhome garden applications for which space availability may be limited.

Fruit size and number. Introduction of presently disclosed transcriptionfactor genes that affect fruit size will have desirable impacts on fruitsize and number, which may comprise increases in yield for fruit crops,or reduced fruit yield, such as when vegetative growth is preferred(e.g., with bushy ornamentals, or where fruit is undesirable, as withornamental olive trees).

Flower structure, inflorescence, and development. Presently disclosedtransgenic transcription factors have been used to create plants withlarger flowers or arrangements of flowers that are distinct fromwild-type or non-transformed cultivars. This would likely have the mostvalue for the ornamental horticulture industry, where larger flowers orinteresting presentations generally are preferred and command thehighest prices. Flower structure may have advantageous effects onfertility, and could be used, for example, to decrease fertility by theabsence, reduction or screening of reproductive components. Oneinteresting application for manipulation of flower structure, forexample, by introduced transcription factors could be in the increasedproduction of edible flowers or flower parts, including saffron, whichis derived from the stigmas of Crocus sativus.

Number and development of trichomes. Several presently disclosedtranscription factor genes have been used to modify trichome number andamount of trichome products in plants. Trichome glands on the surface ofmany higher plants produce and secrete exudates that give protectionfrom the elements and pests such as insects, microbes and herbivores.These exudates may physically immobilize insects and spores, may beinsecticidal or ant-microbial or they may act as allergens or irritantsto protect against herbivores. Trichomes have also been suggested todecrease transpiration by decreasing leaf surface air flow, and byexuding chemicals that protect the leaf from the sun.

Seed size, color and number. The introduction of presently disclosedtranscription factor genes into plants that alter the size or number ofseeds may have a significant impact on yield, both when the product isthe seed itself, or when biomass of the vegetative portion of the plantis increased by reducing seed production. In the case of fruit products,it is often advantageous to modify a plant to have reduced size ornumber of seeds relative to non-transformed plants to provide seedlessor varieties with reduced numbers or smaller seeds. Presently disclosedtranscription factor genes have also been shown to affect seed size,including the development of larger seeds. Seed size, in addition toseed coat integrity, thickness and permeability, seed water content andby a number of other components including antioxidants andoligosaccharides, may affect seed longevity in storage. This would be animportant utility when the seed of a plant is the harvested crops, aswith, for example, peas, beans, nuts, etc. Presently disclosedtranscription factor genes have also been used to modify seed color,which could provide added appeal to a seed product.

Root development, modifications. By modifying the structure ordevelopment of roots by transforming into a plant one or more of thepresently disclosed transcription factor genes, plants may be producedthat have the capacity to thrive in otherwise unproductive soils. Forexample, grape roots that extend further into rocky soils, or thatremain viable in waterlogged soils, would increase the effectiveplanting range of the crop. It may be advantageous to manipulate a plantto produce short roots, as when a soil in which the plant will begrowing is occasionally flooded, or when pathogenic fungi ordisease-causing nematodes are prevalent.

Modifications to root hairs. Presently disclosed transcription factorgenes that increase root hair length or number potentially could be usedto increase root growth or vigor, which might in turn allow better plantgrowth under adverse conditions such as limited nutrient or wateravailability.

Apical dominance. The modified expression of presently disclosedtranscription factors that control apical dominance could be used inornamental horticulture, for example, to modify plant architecture.

Branching patterns. Several presently disclosed transcription factorgenes have been used to manipulate branching, which could providebenefits in the forestry industry. For example, reduction in theformation of lateral branches could reduce knot formation. Conversely,increasing the number of lateral branches could provide utility when aplant is used as a windscreen, or may also provide ornamentaladvantages.

Leaf shape, color and modifications. It has been demonstrated inlaboratory experiments that overexpression of some of the presentlydisclosed transcription factors produced marked effects on leafdevelopment. At early stages of growth, these transgenic seedlingsdeveloped narrow, upward pointing leaves with long petioles, possiblyindicating a disruption in circadian-clock controlled processes ornyctinastic movements. Other transcription factor genes can be used toincrease plant biomass; large size would be useful in crops where thevegetative portion of the plant is the marketable portion.

Siliques. Genes that later silique conformation in brassicates may beused to modify fruit ripening processes in brassicates and other plants,which may positively affect seed or fruit quality.

Stem morphology and shoot modifications. Laboratory studies havedemonstrated that introducing several of the presently disclosedtranscription factor genes into plants can cause stem bifurcations inshoots, in which the shoot meristems split to form two or three separateshoots. This unique appearance would be desirable in ornamentalapplications.

Diseases, pathogens and pests. A number of the presently disclosedtranscription factor genes have been shown to or are likely to conferresistance to various plant diseases, pathogens and pests. The offendingorganisms include fungal pathogens Fusarium oxysporum, Botrytis cinerea,Sclerotinia sclerotiorum, and Erysiphe orontii. Bacterial pathogens towhich resistance may be conferred include Pseudomonas syringae. Otherproblem organisms may potentially include nematodes, mollicutes,parasites, or herbivorous arthropods. In each case, one or moretransformed transcription factor genes may provide some benefit to theplant to help prevent or overcome infestation. The mechanisms by whichthe transcription factors work could include increasing surface waxes oroils, surface thickness, local senescence, or the activation of signaltransduction pathways that regulate plant defense in response to attacksby herbivorous pests (including, for example, protease inhibitors).

Increased tolerance of plants to nutrient-limited soils. Presentlydisclosed transcription factor genes introduced into plants may providethe means to improve uptake of essential nutrients, includingnitrogenous compounds, phosphates, potassium, and trace minerals. Theeffect of these modifications is to increase the seedling germinationand range of ornamental and crop plants. The utilities of presentlydisclosed transcription factor genes conferring tolerance to conditionsof low nutrients also include cost savings to the grower by reducing theamounts of fertilizer needed, environmental benefits of reducedfertilizer runoff; and improved yield and stress tolerance. In addition,this gene could be used to alter seed protein amounts and/or compositionthat could impact yield as well as the nutritional value and productionof various food products.

Hormone sensitivity. One or more of the presently disclosedtranscription factor genes have been shown to affect plant abscisic acid(ABA) sensitivity. This plant hormone is likely the most importanthormone in mediating the adaptation of a plant to stress. For example,ABA mediates conversion of apical meristems into dormant buds. Inresponse to increasingly cold conditions, the newly developing leavesgrowing above the meristem become converted into stiff bud scales thatclosely wrap the meristem and protect it from mechanical damage duringwinter. ABA in the bud also enforces dormancy; during premature warmspells, the buds are inhibited from sprouting. Bud dormancy iseliminated after either a prolonged cold period of cold or a significantnumber of lengthening days. Thus, by affecting ABA sensitivity,introduced transcription factor genes may affect cold sensitivity andsurvivability. ABA is also important in protecting plants from droughttolerance.

Several other of the present transcription factor genes have been usedto manipulate ethylene signal transduction and response pathways. Thesegenes can thus be used to manipulate the processes influenced byethylene, such as seed germination or fruit ripening, and to improveseed or fruit quality.

Production of seed and leaf prenyl lipids, including tocopherol. Prenyllipids play a role in anchoring proteins in membranes or membranousorganelles. Thus, modifying the prenyl lipid content of seeds and leavescould affect membrane integrity and function. A number of presentlydisclosed transcription factor genes have been shown to modify thetocopherol composition of plants. Tocopherols have both anti-oxidant andvitamin E activity.

Production of seed and leaf phytosterols: Presently disclosedtranscription factor genes that modify levels of phytosterols in plantsmay have at least two utilities. First, phytosterols are an importantsource of precursors for the manufacture of human steroid hormones.Thus, regulation of transcription factor expression or activity couldlead to elevated levels of important human steroid precursors forsteroid semi-synthesis. For example, transcription factors that causeelevated levels of campesterol in leaves, or sitosterols andstigmasterols in seed crops, would be useful for this purpose.Phytosterols and their hydrogenated derivatives phytostanols also haveproven cholesterol-lowering properties, and transcription factor genesthat modify the expression of these compounds in plants would thusprovide health benefits.

Production of seed and leaf glucosinolates. Some glucosinolates haveanti-cancer activity; thus, increasing the levels or composition ofthese compounds by introducing several of the presently disclosedtranscription factors might be of interest from a nutraceuticalstandpoint. (3) Glucosinolates form part of a plants natural defenseagainst insects. Modification of glucosinolate composition or quantitycould therefore afford increased protection from predators. Furthermore,in edible crops, tissue specific promoters might be used to ensure thatthese compounds accumulate specifically in tissues, such as theepidermis, which are not taken for consumption.

Modified seed oil content. The composition of seeds, particularly withrespect to seed oil amounts and/or composition, is very important forthe nutritional value and production of various food and feed products.Several of the presently disclosed transcription factor genes in seedlipid saturation that alter seed oil content could be used to improvethe heat stability of oils or to improve the nutritional quality of seedoil, by, for example, reducing the number of calories in seed,increasing the number of calories in animal feeds, or altering the ratioof saturated to unsaturated lipids comprising the oils.

Seed and leaf fatty acid composition. A number of the presentlydisclosed transcription factor genes have been shown to alter the fattyacid composition in plants, and seeds in particular. This modificationmay find particular value for improving the nutritional value of, forexample, seeds or whole plants. Dietary fatty acids ratios have beenshown to have an effect on, for example, bone integrity and remodeling(see, for example, Weiler, H. A., Pediatr Res (2000) 47:5 692–697). Theratio of dietary fatty acids may alter the precursor pools of long-chainpolyunsaturated fatty acids that serve as precursors for prostaglandinsynthesis. In mammalian connective tissue, prostaglandins serve asimportant signals regulating the balance between resorption andformation in bone and cartilage. Thus dietary fatty acid ratios alteredin seeds may affect the etiology and outcome of bone loss.

Modified seed protein content. As with seed oils, the composition ofseeds, particularly with respect to protein amounts and/or composition,is very important for the nutritional value and production of variousfood and feed products. A number of the presently disclosedtranscription factor genes modify the protein concentrations in seedswould provide nutritional benefits, and may be used to prolong storage,increase seed pest or disease resistance, or modify germination rates.

Production of flavonoids in leaves and other plant parts. Expression ofpresently disclosed transcription factor genes that increase flavonoidproduction in plants, including anthocyanins and condensed tannins, maybe used to alter in pigment production for horticultural purposes, andpossibly increasing stress resistance. Flavonoids have antimicrobialactivity and could be used to engineer pathogen resistance. Severalflavonoid compounds have health promoting effects such as the inhibitionof tumor growth and cancer, prevention of bone loss and the preventionof the oxidation of lipids. Increasing levels of condensed tannins,whose biosynthetic pathway is shared with anthocyanin biosynthesis, inforage legumes is an important agronomic trait because they preventpasture bloat by collapsing protein foams within the rumen. For a reviewon the utilities of flavonoids and their derivatives, refer to Dixon etal. (999) Trends Plant Sci. 4:394–400.

Production of diterpenes in leaves and other plant parts. Depending onthe plant species, varying amounts of diverse secondary biochemicals(often lipophilic terpenes) are produced and exuded or volatilized bytrichomes. These exotic secondary biochemicals, which are relativelyeasy to extract because they are on the surface of the leaf, have beenwidely used in such products as flavors and aromas, drugs, pesticidesand cosmetics. Thus, the overexpression of genes that are used toproduce diterpenes in plants may be accomplished by introducingtranscription factor genes that induce said overexpression. One class ofsecondary metabolites, the diterpenes, can effect several biologicalsystems such as tumor progression, prostaglandin synthesis and tissueinflammation. In addition, diterpenes can act as insect pheromones,termite allomones, and can exhibit neurotoxic, cytotoxic and antimitoticactivities. As a result of this functional diversity, diterpenes havebeen the target of research several pharmaceutical ventures. In mostcases where the metabolic pathways are impossible to engineer,increasing trichome density or size on leaves may be the only way toincrease plant productivity.

Production of anthocyanin in leaves and other plant parts. Severalpresently disclosed transcription factor genes can be used to alteranthocyanin production in numerous plant species. The potentialutilities of these genes include alterations in pigment production forhorticultural purposes, and possibly increasing stress resistance incombination with another transcription factor.

Production of miscellaneous secondary metabolites. Microarray datasuggests that flux through the aromatic amino acid biosynthetic pathwaysand primary and secondary metabolite biosynthetic pathways areup-regulated. Presently disclosed transcription factors have been shownto be involved in regulating alkaloid biosynthesis, in part byup-regulating the enzymes indole-3-glycerol phosphatase andstrictosidine synthase. Phenylalanine ammonia lyase, chalcone synthaseand trans-cinnamate mono-oxygenase are also induced, and are involved inphenylpropenoid biosynthesis.

Sugar, starch, hemicellulose composition. Overexpression of thepresently disclosed transcription factors that affect sugar contentresulted in plants with altered leaf insoluble sugar content.Transcription factors that alter plant cell wall composition haveseveral potential applications including altering food digestibility,plant tensile strength, wood quality, pathogen resistance and in pulpproduction. The potential utilities of a gene involved inglucose-specific sugar sensing are to alter energy balance,photosynthetic rate, carbohydrate accumulation, biomass production,source-sink relationships, and senescence.

Hemicellulose is not desirable in paper pulps because of its lack ofstrength compared with cellulose. Thus modulating the amounts ofcellulose vs. hemicellulose in the plant cell wall is desirable for thepaper/lumber industry. Increasing the insoluble carbohydrate content invarious fruits, vegetables, and other edible consumer products willresult in enhanced fiber content. Increased fiber content would not onlyprovide health benefits in food products, but might also increasedigestibility of forage crops. In addition, the hemicellulose and pectincontent of fruits and berries affects the quality of jam and catsup madefrom them. Changes in hemicellulose and pectin content could result in asuperior consumer product.

Plant response to sugars and sugar composition. In addition to theirimportant role as an energy source and structural component of the plantcell, sugars are central regulatory molecules that control severalaspects of plant physiology, metabolism and development. It is thoughtthat this control is achieved by regulating gene expression and, inhigher plants, sugars have been shown to repress or activate plant genesinvolved in many essential processes such as photosynthesis, glyoxylatemetabolism, respiration, starch and sucrose synthesis and degradation,pathogen response, wounding response, cell cycle regulation,pigmentation, flowering and senescence. The mechanisms by which sugarscontrol gene expression are not understood.

Because sugars are important signaling molecules, the ability to controleither the concentration of a signaling sugar or how the plant perceivesor responds to a signaling sugar could be used to control plantdevelopment, physiology or metabolism. For example, the flux of sucrose(a disaccharide sugar used for systemically transporting carbon andenergy in most plants) has been shown to affect gene expression andalter storage compound accumulation in seeds. Manipulation of thesucrose signaling pathway in seeds may therefore cause seeds to havemore protein, oil or carbohydrate, depending on the type ofmanipulation. Similarly, in tubers, sucrose is converted to starch whichis used as an energy store. It is thought that sugar signaling pathwaysmay partially determine the levels of starch synthesized in the tubers.The manipulation of sugar signaling in tubers could lead to tubers witha higher starch content.

Thus, the presently disclosed transcription factor genes that manipulatethe sugar signal transduction pathway may lead to altered geneexpression to produce plants with desirable traits. In particular,manipulation of sugar signal transduction pathways could be used toalter source-sink relationships in seeds, tubers, roots and otherstorage organs leading to increase in yield.

Plant growth rate and development. A number of the presently disclosedtranscription factor genes have been shown to have significant effectson plant growth rate and development. These observations have included,for example, more rapid or delayed growth and development ofreproductive organs. This would provide utility for regions with shortor long growing seasons, respectively. Accelerating plant growth wouldalso improve early yield or increase biomass at an earlier stage, whensuch is desirable (for example, in producing forestry products).

Embryo development. Presently disclosed transcription factor genes thatalter embryo development has been used to alter seed protein and oilamounts and/or composition which is very important for the nutritionalvalue and production of various food products. Seed shape and seed coatmay also be altered by these genes, which may provide for improvedstorage stability.

Seed germination rate. A number of the presently disclosed transcriptionfactor genes have been shown to modify seed germination rate, includingwhen the seeds are in conditions normally unfavorable for germination(e.g., cold, heat or salt stress, or in the presence of ABA), and maythus be used to modify and improve germination rates under adverseconditions.

Plant, seedling vigor. Seedlings transformed with presently disclosedtranscription factors have been shown to possess larger cotyledons andappeared somewhat more advanced than control plants. This indicates thatthe seedlings developed more rapidly that the control plants. Rapidseedling development is likely to reduce loss due to diseasesparticularly prevalent at the seedling stage (e.g., damping off) and isthus important for survivability of plants germinating in the field orin controlled environments.

Senescence, cell death. Presently disclosed transcription factor genesmay be used to alter senescence responses in plants. Although leafsenescence is thought to be an evolutionary adaptation to recyclenutrients, the ability to control senescence in an agricultural settinghas significant value. For example, a delay in leaf senescence in somemaize hybrids is associated with a significant increase in yields and adelay of a few days in the senescence of soybean plants can have a largeimpact on yield. Delayed flower senescence may also generate plants thatretain their blossoms longer and this may be of potential interest tothe ornamental horticulture industry.

Modified fertility. Plants that overexpress a number of the presentlydisclosed transcription factor genes have been shown to possess reducedfertility. This could be a desirable trait, as it could be exploited toprevent or minimize the escape of the pollen of genetically modifiedorganisms (GMOs) into the environment.

Early and delayed flowering. Presently disclosed transcription factorgenes that accelerate flowering could have valuable applications in suchprograms since they allow much faster generation times. In a number ofspecies, for example, broccoli, cauliflower, where the reproductiveparts of the plants constitute the crop and the vegetative tissues arediscarded, it would be advantageous to accelerate time to flowering.Accelerating flowering could shorten crop and tree breeding programs.Additionally, in some instances, a faster generation time might allowadditional harvests of a crop to be made within a given growing season.A number of Arabidopsis genes have already been shown to accelerateflowering when constitutively expressed. These include LEAFY, APETALA1and CONSTANS (Mandel, M. et al., 1995, Nature 377, 522–524; Weigel, D.and Nilsson, O., 1995, Nature 377, 495–500; Simon et al., 1996, Nature384, 59–62).

By regulating the expression of potential flowering using induciblepromoters, flowering could be triggered by application of an inducerchemical. This would allow flowering to be synchronized across a cropand facilitate more efficient harvesting. Such inducible systems couldalso be used to tune the flowering of crop varieties to differentlatitudes. At present, species such as soybean and cotton are availableas a series of maturity groups that are suitable for different latitudeson the basis of their flowering time (which is governed by day-length).A system in which flowering could be chemically controlled would allow asingle high-yielding northern maturity group to be grown at anylatitude. In southern regions such plants could be grown for longer,thereby increasing yields, before flowering was induced. In morenorthern areas, the induction would be used to ensure that the cropflowers prior to the first winter frosts.

In a sizeable number of species, for example, root crops, where thevegetative parts of the plants constitute the crop and the reproductivetissues are discarded, it would be advantageous to delay or preventflowering. Extending vegetative development with presently disclosedtranscription factor genes could thus bring about large increases inyields. Prevention of flowering might help maximize vegetative yieldsand prevent escape of genetically modified organism (GMO) pollen.

Extended flowering phase. Presently disclosed transcription factors thatextend flowering time have utility in engineering plants withlonger-lasting flowers for the horticulture industry, and for extendingthe time in which the plant is fertile.

Flower and leaf development. Presently disclosed transcription factorgenes have been used to modify the development of flowers and leaves.This could be advantageous in the development of new ornamentalcultivars that present unique configurations. In addition, some of thesegenes have been shown to reduce a plant's fertility, which is alsouseful for helping to prevent development of pollen of GMOs.

Flower abscission. Presently disclosed transcription factor genesintroduced into plants have been used to retain flowers for longerperiods. This would provide a significant benefit to the ornamentalindustry, for both cut flowers and woody plant varieties (of, forexample, maize), as well as have the potential to lengthen the fertileperiod of a plant, which could positively impact yield and breedingprograms.

A listing of specific effects and utilities that the presently disclosedtranscription factor genes have on plants, as determined by directobservation and assay analysis, is provided in Table 4.

Antisense and Co-suppression

In addition to expression of the nucleic acids of the invention as genereplacement or plant phenotype modification nucleic acids, the nucleicacids are also useful for sense and anti-sense suppression ofexpression, e.g., to down-regulate expression of a nucleic acid of theinvention, e.g., as a further mechanism for modulating plant phenotype.That is, the nucleic acids of the invention, or subsequences oranti-sense sequences thereof, can be used to block expression ofnaturally occurring homologous nucleic acids. A variety of sense andanti-sense technologies are known in the art, e.g., as set forth inLichtenstein and Nellen (1997) Antisense Technology: A PracticalApproach IRL Press at Oxford University Press, Oxford, U.K. In general,sense or anti-sense sequences are introduced into a cell, where they areoptionally amplified, e.g., by transcription. Such sequences includeboth simple oligonucleotide sequences and catalytic sequences such asribozymes.

For example, a reduction or elimination of expression (i.e., a“knock-out”) of a transcription factor or transcription factor homologuepolypeptide in a transgenic plant, e.g., to modify a plant trait, can beobtained by introducing an antisense construct corresponding to thepolypeptide of interest as a cDNA. For antisense suppression, thetranscription factor or homologue cDNA is arranged in reverseorientation (with respect to the coding sequence) relative to thepromoter sequence in the expression vector. The introduced sequence neednot be the full length cDNA or gene, and need not be identical to thecDNA or gene found in the plant type to be transformed. Typically, theantisense sequence need only be capable of hybridizing to the targetgene or RNA of interest. Thus, where the introduced sequence is ofshorter length, a higher degree of homology to the endogenoustranscription factor sequence will be needed for effective antisensesuppression. While antisense sequences of various lengths can beutilized, preferably, the introduced antisense sequence in the vectorwill be at least 30 nucleotides in length, and improved antisensesuppression will typically be observed as the length of the antisensesequence increases. Preferably, the length of the antisense sequence inthe vector will be greater than 100 nucleotides. Transcription of anantisense construct as described results in the production of RNAmolecules that are the reverse complement of mRNA molecules transcribedfrom the endogenous transcription factor gene in the plant cell.

Suppression of endogenous transcription factor gene expression can alsobe achieved using a ribozyme. Ribozymes are RNA molecules that possesshighly specific endoribonuclease activity. The production and use ofribozymes are disclosed in U.S. Pat. No. 4,987,071 and U.S. Pat. No.5,543,508. Synthetic ribozyme sequences including antisense RNAs can beused to confer RNA cleaving activity on the antisense RNA, such thatendogenous mRNA molecules that hybridize to the antisense RNA arecleaved, which in turn leads to an enhanced antisense inhibition ofendogenous gene expression.

Vectors in which RNA encoded by a transcription factor or transcriptionfactor homologue cDNA is over-expressed can also be used to obtainco-suppression of a corresponding endogenous gene, e.g., in the mannerdescribed in U.S. Pat. No. 5,231,020 to Jorgensen. Such co-suppression(also termed sense suppression) does not require that the entiretranscription factor cDNA be introduced into the plant cells, nor doesit require that the introduced sequence be exactly identical to theendogenous transcription factor gene of interest. However, as withantisense suppression, the suppressive efficiency will be enhanced asspecificity of hybridization is increased, e.g., as the introducedsequence is lengthened, and/or as the sequence similarity between theintroduced sequence and the endogenous transcription factor gene isincreased.

Vectors expressing an untranslatable form of the transcription factormRNA, e.g., sequences comprising one or more stop codon, or nonsensemutation) can also be used to suppress expression of an endogenoustranscription factor, thereby reducing or eliminating its activity andmodifying one or more traits. Methods for producing such constructs aredescribed in U.S. Pat. No. 5,583,021. Preferably, such constructs aremade by introducing a premature stop codon into the transcription factorgene. Alternatively, a plant trait can be modified by gene silencingusing double-strand RNA (Sharp (1999) Genes and Development 13:139–141). Another method for abolishing the expression of a gene is byinsertion mutagenesis using the T-DNA of Agrobacterium tumefaciens.After generating the insertion mutants, the mutants can be screened toidentify those containing the insertion in a transcription factor ortranscription factor homologue gene. Plants containing a singletransgene insertion event at the desired gene can be crossed to generatehomozygous plants for the mutation. Such methods are well known to thoseof skill in the art. (See for example Koncz et al. (1992) Methods inArabidopsis Research, World Scientific.)

Alternatively, a plant phenotype can be altered by, eliminating anendogenous gene, such as a transcription factor or transcription factorhomologue, e.g., by homologous recombination (Kempin et al. (1997)Nature 389:802–803).

A plant trait can also be modified by using the Cre-lox system (forexample, as described in U.S. Pat. No. 5,658,772). A plant genome can bemodified to include first and second lox sites that are then contactedwith a Cre recombinase. If the lox sites are in the same orientation,the intervening DNA sequence between the two sites is excised. If thelox sites are in the opposite orientation, the intervening sequence isinverted.

The polynucleotides and polypeptides of this invention can also beexpressed in a plant in the absence of an expression cassette bymanipulating the activity or expression level of the endogenous gene byother means. For example, by ectopically expressing a gene by T-DNAactivation tagging (Ichikawa et al. (1997) Nature 390 698–701; Kakimotoet al. (1996) Science 274: 982–985). This method entails transforming aplant with a gene tag containing multiple transcriptional enhancers andonce the tag has inserted into the genome, expression of a flanking genecoding sequence becomes deregulated. In another example, thetranscriptional machinery in a plant can be modified so as to increasetranscription levels of a polynucleotide of the invention (See, e.g.,PCT Publications WO 96/06166 and WO 98/53057 which describe themodification of the DNA-binding specificity of zinc finger proteins bychanging particular amino acids in the DNA-binding motif).

The transgenic plant can also include the machinery necessary forexpressing or altering the activity of a polypeptide encoded by anendogenous gene, for example by altering the phosphorylation state ofthe polypeptide to maintain it in an activated state.

Transgenic plants (or plant cells, or plant explants, or plant tissues)incorporating the polynucleotides of the invention and/or expressing thepolypeptides of the invention can be produced by a variety of wellestablished techniques as described above. Following construction of avector, most typically an expression cassette, including apolynucleotide, e.g., encoding a transcription factor or transcriptionfactor homologue, of the invention, standard techniques can be used tointroduce the polynucleotide into a plant, a plant cell, a plant explantor a plant tissue of interest. Optionally, the plant cell, explant ortissue can be regenerated to produce a transgenic plant.

The plant can be any higher plant, including gymnosperms,monocotyledonous and dicotyledenous plants. Suitable protocols areavailable for Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae(carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed,broccoli, etc.), Curcurbitaceae (melons and cucumber), Gramineae (wheat,corn, rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco,peppers, etc.), and various other crops. See protocols described inAmmirato et al. (1984) Handbook of Plant Cell Culture-Crop Species,Macmillan Publ. Co. Shimamoto et al. (1989) Nature 338:274–276; Fromm etal. (1990) Bio/Technology 8:833–839; and Vasil et al. (1990)Bio/Technology 8:429–434.

Transformation and regeneration of both monocotyledonous anddicotyledonous plant cells is now routine, and the selection of the mostappropriate transformation technique will be determined by thepractitioner. The choice of method will vary with the type of plant tobe transformed; those skilled in the art will recognize the suitabilityof particular methods for given plant types. Suitable methods caninclude, but are not limited to: electroporation of plant protoplasts;liposome-mediated transformation; polyethylene glycol (PEG) mediatedtransformation; transformation using viruses; micro-injection of plantcells; micro-projectile bombardment of plant cells; vacuum infiltration;and Agrobacterium tumefaciens mediated transformation. Transformationmeans introducing a nucleotide sequence into a plant in a manner tocause stable or transient expression of the sequence.

Successful examples of the modification of plant characteristics bytransformation with cloned sequences which serve to illustrate thecurrent knowledge in this field of technology, and which are hereinincorporated by reference, include: U.S. Pat. Nos. 5,571,706; 5,677,175;5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526;5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.

Following transformation, plants are preferably selected using adominant selectable marker incorporated into the transformation vector.Typically, such a marker will confer antibiotic or herbicide resistanceon the transformed plants, and selection of transformants can beaccomplished by exposing the plants to appropriate concentrations of theantibiotic or herbicide.

After transformed plants are selected and grown to maturity, thoseplants showing a modified trait are identified. The modified trait canbe any of those traits described above. Additionally, to confirm thatthe modified trait is due to changes in expression levels or activity ofthe polypeptide or polynucleotide of the invention can be determined byanalyzing mRNA expression using Northern blots, RT-PCR or microarrays,or protein expression using immunoblots or Western blots or gel shiftassays.

Integrated Systems—Sequence Identity

Additionally, the present invention may be an integrated system,computer or computer readable medium that comprises an instruction setfor determining the identity of one or more sequences in a database. Inaddition, the instruction set can be used to generate or identifysequences that meet any specified criteria. Furthermore, the instructionset may be used to associate or link certain functional benefits, suchimproved characteristics, with one or more identified sequence.

For example, the instruction set can include, e.g., a sequencecomparison or other alignment program, e.g., an available program suchas, for example, the Wisconsin Package Version 10.0, such as BLAST,FASTA, PILEUP, FINDPATTERNS or the like (GCG, Madison, Wis.). Publicsequence databases such as GenBank, EMBL, Swiss-Prot and PIR or privatesequence databases such as PHYTOSEQ sequence database (Incyte Genomics,Palo Alto, Calif.) can be searched.

Alignment of sequences for comparison can be conducted by the localhomology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482,by the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48:443–453, by the search for similarity method of Pearsonand Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444–2448, bycomputerized implementations of these algorithms. After alignment,sequence comparisons between two (or more) polynucleotides orpolypeptides are typically performed by comparing sequences of the twosequences over a comparison window to identify and compare local regionsof sequence similarity. The comparison window can be a segment of atleast about 20 contiguous positions, usually about 50 to about 200, moreusually about 100 to about 150 contiguous positions. A description ofthe method is provided in Ausubel et al., supra.

A variety of methods for determining sequence relationships can be used,including manual alignment and computer assisted sequence alignment andanalysis. This later approach is a preferred approach in the presentinvention, due to the increased throughput afforded by computer assistedmethods. As noted above, a variety of computer programs for performingsequence alignment are available, or can be produced by one of skill.

One example algorithm that is suitable for determining percent sequenceidentity and sequence similarity is the BLAST algorithm, which isdescribed in Altschul et al. J. Mol. Biol 215:403–410 (1990). Softwarefor performing BLAST analyses is publicly available, e.g., through theNational Center for Biotechnology Information (see internet website atncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915). Unless otherwise indicated, “sequenceidentity” here refers to the % sequence identity generated from atblastx using the NCBI version of the algorithm at the default settingsusing gapped alignments with the filter “off” (see, for example,internet website at ncbi.nlm.nih.gov).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873–5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence (and, therefore, in thiscontext, homologous) if the smallest sum probability in a comparison ofthe test nucleic acid to the reference nucleic acid is less than about0.1, or less than about 0.01, and or even less than about 0.001. Anadditional example of a useful sequence alignment algorithm is PILEUP.PILEUP creates a multiple sequence alignment from a group of relatedsequences using progressive, pairwise alignments. The program can align,e.g., up to 300 sequences of a maximum length of 5,000 letters.

The integrated system, or computer typically includes a user inputinterface allowing a user to selectively view one or more sequencerecords corresponding to the one or more character strings, as well asan instruction set which aligns the one or more character strings witheach other or with an additional character string to identify one ormore region of sequence similarity. The system may include a link of oneor more character strings with a particular phenotype or gene function.Typically, the system includes a user readable output element thatdisplays an alignment produced by the alignment instruction set.

The methods of this invention can be implemented in a localized ordistributed computing environment. In a distributed environment, themethods may implemented on a single computer comprising multipleprocessors or on a multiplicity of computers. The computers can belinked, e.g. through a common bus, but more preferably the computer(s)are nodes on a network. The network can be a generalized or a dedicatedlocal or wide-area network and, in certain preferred embodiments, thecomputers may be components of an intra-net or an internet.

Thus, the invention provides methods for identifying a sequence similaror homologous to one or more polynucleotides as noted herein, or one ormore target polypeptides encoded by the polynucleotides, or otherwisenoted herein and may include linking or associating a given plantphenotype or gene function with a sequence. In the methods, a sequencedatabase is provided (locally or across an inter or intra net) and aquery is made against the sequence database using the relevant sequencesherein and associated plant phenotypes or gene functions.

Any sequence herein can be entered into the database, before or afterquerying the database. This provides for both expansion of the databaseand, if done before the querying step, for insertion of controlsequences into the database. The control sequences can be detected bythe query to ensure the general integrity of both the database and thequery. As noted, the query can be performed using a web browser basedinterface. For example, the database can be a centralized publicdatabase such as those noted herein, and the querying can be done from aremote terminal or computer across an internet or intranet.

EXAMPLES

The following examples are intended to illustrate but not limit thepresent invention. The complete descriptions of the traits associatedwith each polynucleotide of the invention is fully disclosed in Table 4and Table 6.

Example I

Full Length Gene Identification and Cloning

Putative transcription factor sequences (genomic or ESTs) related toknown transcription factors were identified in the Arabidopsis thalianaGenBank database using the tblastn sequence analysis program usingdefault parameters and a P-value cutoff threshold of −4 or −5 or lower,depending on the length of the query sequence. Putative transcriptionfactor sequence hits were then screened to identify those containingparticular sequence strings. If the sequence hits contained suchsequence strings, the sequences were confirmed as transcription factors.

Alternatively, Arabidopsis thaliana cDNA libraries derived fromdifferent tissues or treatments, or genomic libraries were screened toidentify novel members of a transcription family using a low stringencyhybridization approach. Probes were synthesized using gene specificprimers in a standard PCR reaction (annealing temperature 60° C.) andlabeled with ³²P dCTP using the High Prime DNA Labeling Kit (BoehringerMannheim). Purified radiolabelled probes were added to filters immersedin Church hybridization medium (0.5 M NaPO₄ pH 7.0, 7% SDS, 1% w/vbovine serum albumin) and hybridized overnight at 60° C. with shaking.Filters were washed two times for 45 to 60 minutes with 1×SCC, 1% SDS at60° C.

To identify additional sequence 5′ or 3′ of a partial cDNA sequence in acDNA library, 5′ and 3′ rapid amplification of cDNA ends (RACE) wasperformed using the Marathon™ cDNA amplification kit (Clontech, PaloAlto, Calif.). Generally, the method entailed first isolating poly(A)mRNA, performing first and second strand cDNA synthesis to generatedouble stranded cDNA, blunting cDNA ends, followed by ligation of theMarathon™ Adaptor to the cDNA to form a library of adaptor-ligated dscDNA.

Gene-specific primers were designed to be used along with adaptorspecific primers for both 5′ and 3′ RACE reactions. Nested primers,rather than single primers, were used to increase PCR specificity. Using5′ and 3′ RACE reactions, 5′ and 3′ RACE fragments were obtained,sequenced and cloned. The process can be repeated until 5′ and 3′ endsof the full-length gene were identified. Then the full-length cDNA wasgenerated by PCR using primers specific to 5′ and 3′ ends of the gene byend-to-end PCR.

Example II

Construction of Expression Vectors

The sequence was amplified from a genomic or cDNA library using primersspecific to sequences upstream and downstream of the coding region. Theexpression vector was pMEN20 or pMEN65, which are both derived frompMON3 16 (Sanders et al, (1987) Nucleic Acids Research 15:1543–1558) andcontain the CaMV 35S promoter to express transgenes. To clone thesequence into the vector, both pMEN20 and the amplified DNA fragmentwere digested separately with SalI and NotI restriction enzymes at 37°C. for 2 hours. The digestion products were subject to electrophoresisin a 0.8% agarose gel and visualized by ethidium bromide staining. TheDNA fragments containing the sequence and the linearized plasmid wereexcised and purified by using a Qiaquick gel extraction kit (Qiagen,Valencia Calif.). The fragments of interest were ligated at a ratio of3:1 (vector to insert). Ligation reactions using T4 DNA ligase (NewEngland Biolabs, Beverly Mass.) were carried out at 16° C. for 16 hours.The ligated DNAs were transformed into competent cells of the E. colistrain DH5alpha by using the heat shock method. The transformations wereplated on LB plates containing 50 mg/l kanamycin (Sigma, St. Louis,Mo.). Individual colonies were grown overnight in five milliliters of LBbroth containing 50 mg/l kanamycin at 37° C. Plasmid DNA was purified byusing Qiaquick Mini Prep kits (Qiagen).

Example III

Transformation of Agrobacterium with the Expression Vector

After the plasmid vector containing the gene was constructed, the vectorwas used to transform Agrobacterium tumefaciens cells expressing thegene products. The stock of Agrobacterium tumefaciens cells fortransformation were made as described by Nagel et al. (1990) FEMSMicrobiol Letts. 67: 325–328. Agrobacterium strain ABI was grown in 250ml LB medium (Sigma) overnight at 28° C. with shaking until anabsorbance (A₆₀₀) of 0.5–1.0 was reached. Cells were harvested bycentrifugation at 4,000×g for 15 min at 4° C. Cells were thenresuspended in 250 μl chilled buffer (1 mM HEPES, pH adjusted to 7.0with KOH). Cells were centrifuged again as described above andresuspended in 125 μl chilled buffer. Cells were then centrifuged andresuspended two more times in the same HEPES buffer as described aboveat a volume of 100 μl and 750 μl, respectively. Resuspended cells werethen distributed into 40 μl aliquots, quickly frozen in liquid nitrogen,and stored at −80° C.

Agrobacterium cells were transformed with plasmids prepared as describedabove following the protocol described by Nagel et al. For each DNAconstruct to be transformed, 50–100 ng DNA (generally resuspended in 10mM Tris-HCl, 1 mM EDTA, pH 8.0) was mixed with 40 μl of Agrobacteriumcells. The DNA/cell mixture was then transferred to a chilled cuvettewith a 2 mm electrode gap and subject to a 2.5 kV charge dissipated at25 μF and 200 μF using a Gene Pulser II apparatus (Bio-Rad, Hercules,Calif.). After electroporation, cells were immediately resuspended in1.0 ml LB and allowed to recover without antibiotic selection for 2–4hours at 28° C. in a shaking incubator. After recovery, cells wereplated onto selective medium of LB broth containing 100 μg/mlspectinomycin (Sigma) and incubated for 24–48 hours at 28° C. Singlecolonies were then picked and inoculated in fresh medium. The presenceof the plasmid construct was verified by PCR amplification and sequenceanalysis.

Example IV

Transformation of Arabidopsis Plants with Agrobacterium tumefaciens withExpression Vector

After transformation of Agrobacterium tumefaciens with plasmid vectorscontaining the gene, single Agrobacterium colonies were identified,propagated, and used to transform Arabidopsis plants. Briefly, 500 mlcultures of LB medium containing 50 mg/l kanamycin were inoculated withthe colonies and grown at 28° C. with shaking for 2 days until anoptical absorbance at 600 nm wavelength over 1 cm (A₆₀₀) of >2.0 isreached. Cells were then harvested by centrifugation at 4,000×g for 10min, and resuspended in infiltration medium (1/2× Murashige and Skoogsalts (Sigma), 1× Gamborg's B-5 vitamins (Sigma), 5.0% (w/v) sucrose(Sigma), 0.044 μM benzylamino purine (Sigma), 200 μl/l Silwet L-77(Lehle Seeds) until an A₆₀₀ of 0.8 was reached.

Prior to transformation, Arabidopsis thaliana seeds (ecotype Columbia)were sown at a density of ˜10 plants per 4″ pot onto Pro-Mix BX pottingmedium (Hummert International) covered with fiberglass mesh (18 mm×16mm). Plants were grown under continuous illumination (50–75 μE/m²/sec)at 22–23° C. with 65–70% relative humidity. After about 4 weeks, primaryinflorescence stems (bolts) are cut off to encourage growth of multiplesecondary bolts. After flowering of the mature secondary bolts, plantswere prepared for transformation by removal of all siliques and openedflowers.

The pots were then immersed upside down in the mixture of Agrobacteriuminfiltration medium as described above for 30 sec, and placed on theirsides to allow draining into a 1′×2′ flat surface covered with plasticwrap. After 24 h, the plastic wrap was removed and pots are turnedupright. The immersion procedure was repeated one week later, for atotal of two immersions per pot. Seeds were then collected from eachtransformation pot and analyzed following the protocol described below.

Example V

Identification of Arabidopsis Primary Transformants

Seeds collected from the transformation pots were sterilized essentiallyas follows. Seeds were dispersed into in a solution containing 0.1%(v/v) Triton X-100 (Sigma) and sterile H₂O and washed by shaking thesuspension for 20 min. The wash solution was then drained and replacedwith fresh wash solution to wash the seeds for 20 min with shaking.After removal of the second wash solution, a solution containing 0.1%(v/v) Triton X-100 and 70% ethanol (Equistar) was added to the seeds andthe suspension was shaken for 5 min. After removal of theethanol/detergent solution, a solution containing 0.1% (v/v) TritonX-100 and 30% (v/v) bleach (Clorox) was added to the seeds, and thesuspension was shaken for 10 min. After removal of the bleach/detergentsolution, seeds were then washed five times in sterile distilled H₂O.The seeds were stored in the last wash water at 4° C. for 2 days in thedark before being plated onto antibiotic selection medium (1× Murashigeand Skoog salts (pH adjusted to 5.7 with 1M KOH), 1× Gamborg's B-5vitamins, 0.9% phytagar (Life Technologies), and 50 mg/l kanamycin).Seeds were germinated under continuous illumination (50–75 μE/m²/sec) at22–23° C. After 7–10 days of growth under these conditions, kanamycinresistant primary transformants (T₁ generation) were visible andobtained. These seedlings were transferred first to fresh selectionplates where the seedlings continued to grow for 3–5 more days, and thento soil (Pro-Mix BX potting medium).

Primary transformants were crossed and progeny seeds (T₂) collected;kanamycin resistant seedlings were selected and analyzed. The expressionlevels of the recombinant polynucleotides in the transformants variesfrom about a 5% expression level increase to a least a 100% expressionlevel increase. Similar observations are made with respect topolypeptide level expression.

Example VI

Identification of Arabidopsis Plants with Transcription Factor GeneKnockouts

The screening of insertion mutagenized Arabidopsis collections for nullmutants in a known target gene was essentially as described in Krysan etal (1999) Plant Cell 11:2283–2290. Briefly, gene-specific primers,nested by 5–250 base pairs to each other, were designed from the 5′ and3′ regions of a known target gene. Similarly, nested sets of primerswere also created specific to each of the T-DNA or transposon ends (the“right” and “left” borders). All possible combinations of gene specificand T-DNA/transposon primers were used to detect by PCR an insertionevent within or close to the target gene. The amplified DNA fragmentswere then sequenced which allows the precise determination of theT-DNA/transposon insertion point relative to the target gene. Insertionevents within the coding or intervening sequence of the genes weredeconvoluted from a pool comprising a plurality of insertion events to asingle unique mutant plant for functional characterization. The methodis described in more detail in Yu and Adam, U.S. application Ser. No.09/177,733 filed Oct. 23, 1998.

Example VII

Identification of Modified Phenotypes in Overexpression or Gene KnockoutPlants

Experiments were performed to identify those transformants or knockoutsthat exhibited modified biochemical characteristics. Among thebiochemicals that were assayed were insoluble sugars, such as arabinose,fucose, galactose, mannose, rhamnose or xylose or the like; prenyllipids, such as lutein, beta-carotene, xanthophyll-1, xanthophyll-2,chlorophylls A or B, or alpha-, delta- or gamma-tocopherol or the like;fatty acids, such as 16:0 (palmitic acid), 16:1 (palmitoleic acid), 18:0(stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid), 20:0 , 18:3(linolenic acid), 20:1 (eicosenoic acid), 20:2, 22:1 (erucic acid) orthe like; waxes, such as by altering the levels of C29, C31, or C33alkanes; sterols, such as brassicasterol, campesterol, stigmasterol,sitosterol or stigmastanol or the like, glucosinolates, protein or oillevels.

Fatty acids were measured using two methods depending on whether thetissue was from leaves or seeds. For leaves, lipids were extracted andesterified with hot methanolic H₂SO₄ and partitioned into hexane frommethanolic brine. For seed fatty acids, seeds were pulverized andextracted in methanol:heptane:toluene:2,2-dimethoxypropane:H₂SO₄(39:34:20:5:2) for 90 minutes at 80° C. After cooling to roomtemperature the upper phase, containing the seed fatty acid esters, wassubjected to GC analysis. Fatty acid esters from both seed and leaftissues were analyzed with a Supelco SP-2330 column.

Glucosinolates were purified from seeds or leaves by first heating thetissue at 95° C. for 10 minutes. Preheated ethanol:water (50:50) is andafter heating at 95° C. for a further 10 minutes, the extraction solventis applied to a DEAE Sephadex column which had been previouslyequilibrated with 0.5 M pyridine acetate. Desulfoglucosinolates wereeluted with 300 ul water and analyzed by reverse phase HPLC monitoringat 226 nm.

For wax alkanes, samples were extracted using an identical method asfatty acids and extracts were analyzed on a HP 5890 GC coupled with a5973 MSD. Samples were chromatographically isolated on a J&W DB35 massspectrometer (J&W Scientific).

To measure prenyl lipids levels, seeds or leaves were pulverized with 1to 2% pyrogallol as an antioxidant. For seeds, extracted samples werefiltered and a portion removed for tocopherol and carotenoid/chlorophyllanalysis by HPLC. The remaining material was saponified for steroldetermination. For leaves, an aliquot was removed and diluted withmethanol and chlorophyll A, chlorophyll B, and total carotenoidsmeasured by spectrophotometry by determining optical absorbance at 665.2nm, 652.5 nm, and 470 nm. An aliquot was removed for tocopherol andcarotenoid/chlorophyll composition by HPLC using a Waters uBondapak C18column (4.6 mm×150 mm). The remaining methanolic solution was saponifiedwith 10% KOH at 80° C. for one hour. The samples were cooled and dilutedwith a mixture of methanol and water. A solution of 2% methylenechloride in hexane was mixed in and the samples were centrifuged. theaqueous methanol phase was again re-extracted 2% methylene chloride inhexane and, after centrifugation, the two upper phases were combined andevaporated. 2% methylene chloride in hexane was added to the tubes andthe samples were then extracted with one ml of water. The upper phasewas removed, dried, and resuspended in 400 ul of 2% methylene chloridein hexane and analyzed by gas chromatography using a 50 m DB-5ms (0.25mm ID, 0.25 um phase, J&W Scientific).

Insoluble sugar levels were measured by the method essentially describedby Reiter et al. (1999), Plant Journal 12:335–345. This method analyzesthe neutral sugar composition of cell wall polymers found in Arabidopsisleaves. Soluble sugars were separated from sugar polymers by extractingleaves with hot 70% ethanol. The remaining residue containing theinsoluble polysaccharides was then acid hydrolyzed with allose added asan internal standard. Sugar monomers generated by the hydrolysis werethen reduced to the corresponding alditols by treatment with NaBH4, thenwere acetylated to generate the volatile alditol acetates which werethen analyzed by GC-FID. Identity of the peaks was determined bycomparing the retention times of known sugars converted to thecorresponding alditol acetates with the retention times of peaks fromwild-type plant extracts. Alditol acetates were analyzed on a SupelcoSP-2330 capillary column (30 m×250 um×0.2 um) using a temperatureprogram beginning at 180° C. for 2 minutes followed by an increase to220° C. in 4 minutes. After holding at 220° C. for 10 minutes, the oventemperature is increased to 240° C. in 2 minutes and held at thistemperature for 10 minutes and brought back to room temperature.

To identify plants with alterations in total seed oil or proteincontent, 150 mg of seeds from T2 progeny plants were subjected toanalysis by Near Infrared Reflectance Spectroscopy (NIRS) using a FossNirSystems Model 6500 with a spinning cup transport system. NIRS is anon-destructive analytical method used to determine seed oil and proteincomposition. Infrared is the region of the electromagnetic spectrumlocated after the visible region in the direction of longer wavelengths.‘Near infrared’ owns its name for being the infrared region near to thevisible region of the electromagnetic spectrum. For practical purposes,near infrared comprises wavelengths between 800 and 2500 nm. NIRS isapplied to organic compounds rich in O—H bonds (such as moisture,carbohydrates, and fats), C—H bonds (such as organic compounds andpetroleum derivatives), and N—H bonds (such as proteins and aminoacids). The NIRS analytical instruments operate by statisticallycorrelating NIRS signals at several wavelengths with the characteristicor property intended to be measured. All biological substances containthousands of C—H, O—H, and N—H bonds. Therefore, the exposure to nearinfrared radiation of a biological sample, such as a seed, results in acomplex spectrum which contains qualitative and quantitative informationabout the physical and chemical composition of that sample.

The numerical value of a specific analyte in the sample, such as proteincontent or oil content, is mediated by a calibration approach known aschemometrics. Chemometrics applies statistical methods such as multiplelinear regression (MLR), partial least squares (PLS), and principlecomponent analysis (PCA) to the spectral data and correlates them with aphysical property or other factor, that property or factor is directlydetermined rather than the analyte concentration itself The method firstprovides “wet chemistry” data of the samples required to develop thecalibration.

Calibration for Arabidopsis seed oil composition was performed usingaccelerated solvent extraction using 1 g seed sample size and wasvalidated against certified canola seed. A similar wet chemistryapproach was performed for seed protein composition calibration.

Data obtained from NIRS analysis was analyzed statistically using anearest-neighbor (N-N) analysis. The N-N analysis allows removal ofwithin-block spatial variability in a fairly flexible fashion which doesnot require prior knowledge of the pattern of variability in thechamber. Ideally, all hybrids are grown under identical experimentalconditions within a block (rep). In reality, even in many block designs,significant within-block variability exists. Nearest-neighbor proceduresare based on assumption that environmental effect of a plot is closelyrelated to that of its neighbors. Nearest-neighbor methods useinformation from adjacent plots to adjust for within-block heterogeneityand so provide more precise estimates of treatment means anddifferences. If there is within-plot heterogeneity on a spatial scalethat is larger than a single plot and smaller than the entire block,then yields from adjacent plots will be positively correlated.Information from neighboring plots can be used to reduce or remove theunwanted effect of the spatial heterogeneity, and hence improve theestimate of the treatment effect. Data from neighboring plots can alsobe used to reduce the influence of competition between adjacent plots.The Papadakis N-N analysis can be used with designs to removewithin-block variability that would not be removed with the standardsplit plot analysis (Papadakis, 1973, Inst. d'Amelior. PlantesThessaloniki (Greece) Bull. Scientif., No. 23; Papadakis, 1984, Proc.Acad. Athens, 59, 326–342).

Experiments were performed to identify those transformants or knockoutsthat exhibited an improved pathogen tolerance. For such studies, thetransformants were exposed to biotropic fungal pathogens, such asErysiphe orontii, and necrotropic fungal pathogens, such as Fusariumoxysporum. Fusarium oxysporum isolates cause vascular wilts and dampingoff of various annual vegetables, perennials and weeds (Mauch-Mani andSlusarenko (1994) Molecular Plant-Microbe Interactions 7: 378–383). ForFusarium oxysporum experiments, plants grown on Petri dishes weresprayed with a fresh spore suspension of F. oxysporum. The sporesuspension was prepared as follows: A plug of fungal hyphae from a plateculture was placed on a fresh potato dextrose agar plate and allowed tospread for one week. 5 ml sterile water was then added to the plate,swirled, and pipetted into 50 ml Armstrong Fusarium medium. Spores weregrown overnight in Fusarium medium and then sprayed onto plants using aPreval paint sprayer. Plant tissue was harvested and frozen in liquidnitrogen 48 hours post infection.

Erysiphe orontii is a causal agent of powdery mildew. For Erysipheorontii experiments, plants were grown approximately 4 weeks in agreenhouse under 12 hour light (20° C., ˜30% relative humidity (rh)).Individual leaves were infected with E. orontii spores from infectedplants using a camel's hair brush, and the plants were transferred to aPercival growth chamber (20° C., 80% rh.). Plant tissue was harvestedand frozen in liquid nitrogen 7 days post infection.

Botrytis cinerea is a necrotrophic pathogen. Botrytis cinerea was grownon potato dextrose agar in the light. A spore culture was made byspreading 10 ml of sterile water on the fungus plate, swirling andtransferring spores to 10 ml of sterile water. The spore inoculum(approx. 105 spores/ml) was used to spray 10 day-old seedlings grownunder sterile conditions on MS (minus sucrose) media. Symptoms wereevaluated every day up to approximately 1 week.

Infection with bacterial pathogens Pseudomonas syringae pv maculicola(Psm) strain 4326 and pv maculicola strain 4326 was performed by handinoculation at two doses. Two inoculation doses allows thedifferentiation between plants with enhanced susceptibility and plantswith enhanced resistance to the pathogen. Plants were grown for 3 weeksin the greenhouse, then transferred to the growth chamber for theremainder of their growth. Psm ES4326 was hand inoculated with 1 mlsyringe on 3 fully-expanded leaves per plant (4½ wk old), using at least9 plants per overexpressing line at two inoculation doses, OD=0.005 andOD=0.0005. Disease scoring occurred at day 3 post-inoculation withpictures of the plants and leaves taken in parallel.

In some instances, expression patterns of the pathogen-induced genes(such as defense genes) was monitored by microarray experiments. cDNAswere generated by PCR and resuspended at a final concentration of ˜100ng/ul in 3×SSC or 150 mM Na-phosphate (Eisen and Brown (1999) MethodsEnzymol. 303:179–205). The cDNAs were spotted on microscope glass slidescoated with polylysine. The prepared cDNAs were aliquoted into 384 wellplates and spotted on the slides using an x-y-z gantry (OmniGrid)purchased from GeneMachines (Menlo Park, Calif.) outfitted with quilltype pins purchased from Telechem International (Sunnyvale, Calif.).After spotting, the arrays were cured for a minimum of one week at roomtemperature, rehydrated and blocked following the protocol recommendedby Eisen and Brown (1999; supra).

Sample total RNA (10 ug) samples were labeled using fluorescent Cy3 andCy5 dyes. Labeled samples were resuspended in 4×SSC/0.03% SDS/4 ugsalmon sperm DNA/2 ug tRNA/50 mM Na-pyrophosphate, heated for 95° C. for2.5 minutes, spun down and placed on the array. The array was thencovered with a glass coverslip and placed in a sealed chamber. Thechamber was then kept in a water bath at 62° C. overnight. The arrayswere washed as described in Eisen and Brown (1999) and scanned on aGeneral Scanning 3000 laser scanner. The resulting files aresubsequently quantified using Imagene, a software purchased fromBioDiscovery (Los Angeles, Calif.).

Experiments were performed to identify those transformants or knockoutsthat exhibited an improved environmental stress tolerance. For suchstudies, the transformants were exposed to a variety of environmentalstresses. Plants were exposed to chilling stress (6 hour exposure to4–8° C.), heat stress (6 hour exposure to 32–37° C.), high salt stress(6 hour exposure to 200 mM NaCl), drought stress (168 hours afterremoving water from trays), osmotic stress (6 hour exposure to 3 Mmannitol), or nutrient limitation (nitrogen, phosphate, and potassium)(Nitrogen: all components of MS medium remained constant except N wasreduced to 20 mg/l of NH₄NO₃, or Phosphate: All components of MS mediumexcept KH₂PO₄, which was replaced by K₂SO₄, Potassium: All components ofMS medium except removal of KNO₃ and KH₂PO₄, which were replaced byNaH₄PO₄).

Experiments were performed to identify those transformants or knockoutsthat exhibited a modified structure and development characteristics. Forsuch studies, the transformants were observed by eye to identify novelstructural or developmental characteristics associated with the ectopicexpression of the polynucleotides or polypeptides of the invention.

Experiments were performed to identify those transformants or knockoutsthat exhibited modified sugar-sensing. For such studies, seeds fromtransformants were germinated on media containing 5% glucose or 9.4%sucrose which normally partially restrict hypocotyl elongation. Plantswith altered sugar sensing may have either longer or shorter hypocotylsthan normal plants when grown on this media. Additionally, other planttraits may be varied such as root mass.

Flowering time was measured by the number of rosette leaves present whena visible inflorescence of approximately 3 cm is apparent Rosette andtotal leaf number on the progeny stem are tightly correlated with thetiming of flowering (Koornneef et al (1991) Mol. Gen. Genet 229:57–66.The vernalization response was measured. For vernalization treatments,seeds were sown to MS agar plates, sealed with micropore tape, andplaced in a 4° C. cold room with low light levels for 6–8 weeks. Theplates were then transferred to the growth rooms alongside platescontaining freshly sown non-vernalized controls. Rosette leaves werecounted when a visible inflorescence of approximately 3 cm was apparent.

Modified phenotypes observed for particular overexpressor or knockoutplants are provided in Table 4. For a particular overexpressor thatshows a less beneficial characteristic, it may be more useful to selecta plant with a decreased expression of the particular transcriptionfactor. For a particular knockout that shows a less beneficialcharacteristic, it may be more useful to select a plant with anincreased expression of the particular transcription factor.

The sequences of the Sequence Listing or those in Tables 4 or 5 or thosedisclosed here can be used to prepare transgenic plants and plants withaltered traits. The specific transgenic plants listed below are producedfrom the sequences of the Sequence Listing, as noted. Table 4 providesexemplary polynucleotide and polypeptide sequences of the invention.Table 4 includes, from left to right for each sequence: the first columnshows the polynucleotide SEQ ID NO; the second column shows the MendelGene ID No., GID; the third column shows the trait(s) resulting from theknock out or overexpression of the polynucleotide in the transgenicplant; the fourth column shows the category of the trait; the fifthcolumn shows the transcription factor family to which the polynucleotidebelongs; the sixth column (“Comment”), includes specific effects andutilities conferred by the polynucleotide of the first column; theseventh column shows the SEQ ID NO of the polypeptide encoded by thepolynucleotide; and the eighth column shows the amino acid residuepositions of the conserved domain in amino acid (AA) co-ordinates.

G720: The complete sequence of G720 (SEQ ID NO: 25); similar to aportion of APRR2, Arabidopsis pseudo-response regulator (APRR2; Makinoet al. 2000 Plant Cell Physiol. 41:791–803) was determined. A linehomozygous for a T-DNA insertion in G720 and lines overexpressing G720under the 35S promoter were used to determine the function of this gene.The T-DNA insertion in G720 was approximately half-way into the codingsequence, just before the conserved domain, and therefore should resultin a null mutation. G720 knockout mutants were slightly more sensitiveto freezing than the wild-type controls when the seedlings werecold-acclimated prior to freezing. G720 overexpressing lines wereslightly more tolerant to freezing. When seedlings were frozen at −10°C. for 20 hours, the G720 plants recovered slightly better compared tothe wild-type control in two separate experiments. G720 was induced byABA, salt, osmotic stress, drought, heat, and auxin. The combination ofenhanced sensitivity to freezing in the knockout mutants, enhancedresistance in the overexpressing lines, and the induction pattern ofG720 comprised strong evidence that G720 functions in regulation ofdehydration tolerance, as freezing is a form of dehydration stress.

Plants overexpressing G720 also showed reduced time to flowering in theT1 generation. One third of the 35S::G720 T1 seedlings, from each of twoseparate batches, flowered markedly earlier (up to 1 week sooner,24-hour light conditions) than controls plants. All of the T1 linesshowed high levels of G720 overexpression (determined by RT-PCR). Threeearly flowering T1 plants were selected for further study. However, noneof these lines flowered early in the T2, suggesting that activity of thetransgene might have been reduced between the generations.

Closely Related Genes from Other Species

G720 showed significant similarity to a drought-induced M. truncatulaEST, GenBank accession number BG450227, that encodes a pseudo-receiverdomain. The sequence similarity is high enough to suggest that the twoproteins are orthologs, and the fact that G720 was also drought-inducedis consistent with this hypothesis. Other ESTs from tomato and potato(BG642566, BG128919, BG129142, and BG887673) also showed high similarityto G720 and represent potential orthologs.

G1792: G1792 (SEQ ID NO: 45) was studied using transgenic plants inwhich the gene was expressed under the control of the 35S promoter.35S::G1792 plants were more tolerant to the fungal pathogens Fusariumoxysporum and Botrytis cinerea and showed fewer symptoms afterinoculation with a low dose of each pathogen. This result was confirmedusing individual T2 lines. The effect of G1792 overexpression inincreasing tolerance to pathogens received further, incidentalconfirmation. T2 plants of 35S::G1792 lines 5 and 12 had been growing ina room that suffered a serious powdery mildew infection. For each line,a pot of 6 plants was present in a flat containing 9 other pots of linesfrom unrelated genes. In either of the two different flats, the onlyplants that were free from infection were those from the 35S::G1792line. This observation suggests that G1792 overexpression might increaseresistance to powdery mildew. Additional experiments confirmed that35S::G1792 plants showed increased tolerance to Erysiphe. G1792 wasubiquitously expressed, but appears to be induced by salicylic acid.

35S::G1792 overexpressing plants also showed more tolerance to growthunder nitrogen-limiting conditions. In a root growth assay underconditions of limiting N, 35S::G1792 lines were slightly less stunted.In a germination assay that monitors the effect of C on N signalingthrough anthocyanin production on high sucrose plus and minus glutaminethe 35S::G1792 lines make less anthocyanin on high sucrose plusglutamine, suggesting that the gene can be involved in the plantsability to monitor their carbon and nitrogen status.

G1792 overexpressing plants showed several mild morphologicalalterations: leaves were dark green and shiny, and plants bolted,subsequently senesced, slightly later than wild-type controls. Among theT1 plants, additional morphological variation (not reproduced later inthe T2 plants) was observed: many showed reductions in size as well asaberrations in leaf shape, phyllotaxy, and flower development.

Closely Related Genes from Other Species

G1792 shows sequence similarity, outside the conserved AP2 domain, witha portion of a predicted protein from tomato, represented by ESTsequence AI776626 (AI776626 EST257726 tomato resistant, CornellLycopersicon esculentum cDNA clone cLER19A14, mRNA sequence).

G1756: G1756 (SEQ ID NO:175) was studied using transgenic plants inwhich the gene was expressed under the control of the 35S promoter.Overexpression of G1756 caused alterations in plant growth anddevelopment, reducing overall plant size and fertility. In addition,35S::G1756 overexpressing lines show more disease symptoms followinginoculation with a low dose of the fungal pathogen Botrytis cinereacompared to the wild-type controls. G1756 was ubiquitously expressed andtranscript levels were altered by a variety of environmental orphysiological conditions; G1756 expression can be induced by auxin,cold, and Fusarium.

Closely Related Genes from Other Species

G1756 shows some sequence similarity with known genes from other plantspecies within the conserved WRKY domain.

G481: Northern blot data from five different tissue samples indicatesthat G481 (SEQ ID NO: 113) was primarily expressed in flower and/orsilique, and root tissue. G481 was analyzed through its ectopicoverexpression in plants. G481 overexpressors were more tolerant to highsucrose in a germination assay. The phenotype of G481 was mild; however,there was a consistent difference in the hypocotyl and root elongationin the overexpressor plants compared to wild-type controls.Sucrose-sensing has been implicated in the regulation of source-sinkrelationships in plants. Consistent with the sugar sensing phenotype ofthe G481 overexpressors were the results from the biochemical analysisof G481 overexpressor plants suggesting that line 14 had higher amountsof seed oils and lower amounts of seed protein. This suggested that G481was involved in the allocation of storage compounds to the seed. G481overexpressor line 8 was darker green in the T2 generation which canmean a higher photosynthetic rate consistent with the possible role ofG481 in sugar sensing.

Closely Related Genes from Other Species

There are several sequences from higher plants that show significanthomology to G481 including, X59714 from corn, and two ESTs from tomato,AI486503 and AI782351.

G2133: G2133 (SEQ ID NO: 141) was studied using transgenic plants inwhich the gene was expressed under the control of the 35S promoter.Overexpression of G2133 caused a variety of alterations in plant growthand development: delayed flowering, altered inflorescence architecture,and a decrease in overall size and fertility. At early stages,35S::G2133 transformants were markedly smaller than controls anddisplayed curled, dark-green leaves. Most of these plants remained in avegetative phase of development substantially longer than controls, andproduced an increased number of leaves before bolting. In the mostseverely affected plants, bolting occurred more than a month later thanin wild type (24-hour light). In addition, the plants displayed areduction in apical dominance and formed large numbers of shootssimultaneously, from the axils of rosette leaves. These inflorescencestems had short internodes, and carried increased numbers of caulineleaf nodes, giving them a very leafy appearance. The fertility of35S::G2133 plants was generally very low. In addition, G2133overexpressing lines were more resistant to the herbicide glyphosate. Ina repeat experiment, lines 4 and 5 were more tolerant while line 2 waswild-type. G2133 expression was detected in a variety of tissues:flower, leaf, embryo, and silique samples. Its expression was altered byseveral conditions, including auxin treatment, osmotic stress, andFusarium infection. G2133 can be used for the generation of glyphosateresistant plants, and to increase plant resistance to oxidative stress.

Closely Related Genes from Other Species

G2133 shows some sequence similarity with known genes from other plantspecies within the conserved AP2/EREBP domain.

G2517: G2517 (SEQ ID NO: 143) was studied using transgenic plants inwhich the gene was expressed under the control of the 35S promoter.Overexpression of G2517 caused alterations in plant growth anddevelopment: size variation was apparent in the 35S::G2517 T1generation, with at least half the lines being very small. Additionally,4/12 T1 plants formed flower buds marginally earlier than wild type.Three T1 lines (#8,11,12) were examined in the T2 generation, and allthree T2 populations were slightly smaller than controls. In thephysiological analysis of the T2 populations, G2517 overexpressing lineswere more resistant to the herbicide glyphosate. G2517 can be used forthe generation of glyphosate resistant plants, and to increase plantresistance to oxidative stress.

Closely Related Genes from Other Species

G2517 shows some sequence similarity with known genes from other plantspecies within the conserved WRKY domain.

G2140: The complete sequence of G2140 (SEQ ID NO: 81) was determined.G2140 was expressed throughout the plant. It showed repression bysalicylic acid and Erysiphe infection. Overexpressing G2140 inArabidopsis resulted in seedlings that were more tolerant to osmoticstress conditions. In germination assays where seedlings were exposed tohigh concentrations of sucrose or NaCl, all three lines tested showedbetter cotyledon expansion and seedling vigor. Additionally, G2140overexpressing plants showed insensitivity to ABA in a germinationassay. In general, G2140 overexpressing plants were small and sicklywith short roots when grown in Petri plates. The combination of ABAinsensitivity and resistance to osmotic stress at germination had alsobeen observed for other genes, for example, G1820 (SEQ ID NO:13) andG926 (SEQ ID NO:111). Significantly, the ABA resistance was detected ina germination assay. ABA is involved in maintaining seed dormancy, andit is possible that ABA insensitivity at the germination stage promotesgermination despite unfavorable conditions.

When grown in soil, G2140 overexpressing plants displayed marked changesin Arabidopsis leaf and root morphology. All twenty of the 35S::G2140primary transformants displayed, to various extents, leaves withupcurled margins. In the most severe cases, the leaves became highlycontorted and the plants were slightly small and grew more slowly thancontrols. Three T1 lines (#12,15 and 16) that showed substantial levelsof G2140 overexpression (determined by RT-PCR) were chosen for furtherstudy. The T2 seedlings from each of these lines exhibited stunted rootscompared with controls. Seedlings from two of the lines (#15,16) alsoshowed upcurled cotyledons. At later stages, however, T2-16 plantsappeared wild type. Plants from the T2-12 and T2-15 populations wererather varied in size and showed hints of leaf curling later indevelopment. However, this effect was less severe than that seen in theT1 lines. To verify the leaf-curling phenotype, two further T2populations (#3,18) were morphologically examined; seedlings from T2-3were extremely tiny with thickened hypocotyls and short stunted roots.Such plants were too small for transfer to soil. However, T2-18 plantsshowed slightly contorted cotyledons and formed severely upcurledleaves, confirming the effects seen in the T1 generation.

G2140 is useful for creating plants that germinate better underconditions of high salt. Evaporation from the soil surface causes upwardwater movement and salt accumulation in the upper soil layer where theseeds are placed. Thus, germination normally takes place at a saltconcentration much higher than the mean salt concentration in the wholesoil profile. Increased salt tolerance during the germination stage of acrop plant will impact survivability and yield. In addition, G2140 canbe used to alter a plant's response to water deficit conditions and,therefore, can be used to engineer plants with enhanced tolerance todrought, and freezing.

Closely Related Genes from Other Species

G2140 proteins show extensive sequence similarity with a tomato ovarycDNA, TAMU Lycopersicon esculentum (AI488313) and a Glycine max cDNAclone (BE020519).

G1946: G1946 (SEQ ID NO:49) was studied using transgenic plants in whichthe gene was expressed under the control of the 35S promoter.Overexpression of G1946 resulted in accelerated flowering, with35S::G1946 transformants producing flower buds up to a week earlier thanwild-type controls (24-hour light conditions). These effects were seenin 12/20 primary transformants and in two independent plantings of eachof the three T2 lines. Unlike many early flowering Arabidopsistransgenic lines, which are dwarfed, 35S::G1946 transformants oftenreached full-size at maturity, and produced large quantities of seeds,although the plants were slightly pale in coloration and had slightlyflat leaves compared to wild-type. In addition, 35S::G1946 plants showedan altered response to phosphate deprivation. Seedlings of G1946overexpressors showed more secondary root growth on phosphate-freemedia, when compared to wild-type control. In a repeat experiment, allthree lines showed the phenotype. Overexpression of G1946 in Arabidopsisalso resulted in an increase in seed glucosinolate M39501 in T2 lines 1and 3. An increase in seed oil and a decrease in seed protein was alsoobserved in these two lines. G1946 was ubiquitously expressed, and doesnot appear to be significantly induced or repressed by any of the bioticand abiotic stress conditions tested, with the exception of cold, whichrepressed G1946 expression. G1946 can be used to modify flowering time,as well as to improve the plant's performance in conditions of limitedphosphate, and to alter seed oil, protein, and glucosinolatecomposition.

Closely Related Genes from Other Species

A comparison of the amino acid sequence of G1946 with sequencesavailable from GenBank showed strong similarity with plant HSFs ofseveral species (Lycopersicon peruvianum, Medicago truncatula,Lycopersicon esculentum, Glycine max, Solanum tuberosum, Oryza sativaand Hordeum vulgare subsp. Vulgare).

G1852: G1852 (SEQ ID NO:71) was analyzed through its ectopicoverexpression in plants. Analysis of the endogenous level of G1852transcripts by RT-PCR revealed expression in all tissues tested. G1852expression was induced in response to ABA, heat and drought treatment.35S::G1852 overexpressor plants were more tolerant to osmotic stress ina root growth assay on PEG (polyethylene glycol)-containing mediacompared with wild-type controls. Seedlings were slightly larger andhave more root growth. G1852 can be used to alter a plant's response towater deficit conditions and therefore, be used to engineer plants withenhanced tolerance to drought, salt stress, and freezing.

Closely Related Genes from Other Species

A comparison of the amino acid sequence of G1852 with entries availablefrom GenBank shows strong similarity with plant ankyrins of severalspecies (Malus domestica, Solanum tuberosum, Oryza sativa, Gossypiumarboreum, Medicago truncatula, Glycine max, Lycopersicon esculentum,Pinus taeda, Lotus japonicus and Gossypium hirsutum).

G325: G325 (SEQ ID NO:95) was analyzed using transgenic plants in whichG325 was expressed under the control of the 35S promoter. G325overexpressing plants showed more tolerance to osmotic stress in agermination assay in three separate experiments. They showed moreseedling vigor than wild-type control when germinated on platescontaining high salt and high sucrose. G325 was expressed at high levelsin flowers and cauline leaves, and at lower levels in shoots, rosetteleaves, and seedlings. G325 was induced by auxin, cold and heat stress.Expression of G325 was reduced in response to Fusarium infection orsalicylic acid treatment. G325 can be useful for enhancing seedgermination under high salt conditions or other conditions of osmoticstress. Evaporation from the soil surface causes upward water movementand salt accumulation in the upper soil layer where the seeds areplaced. Thus, germination normally takes place at a salt concentrationmuch higher than the mean salt concentration of in the whole soilprofile. Increased salt tolerance during the germination stage of a cropplant will impact survivability and yield. G325 can also be used toengineer plants with enhanced tolerance to drought, salt stress, andfreezing at later stages during growth and development.

Closely Related Genes from Other Species

G325 showed homology to non-Arabidopsis proteins within the conserveddomain.

G2583: G2583 (SEQ ID NO: 17) was studied using transgenic plants inwhich the gene was expressed under the control of the 35S promoter.35S::G2583 plants exhibited extremely glossy leaves. At early stages,35S::G2583 seedlings appeared normal, but by about two weeks aftersowing, the plants exhibited very striking shiny leaves, which wereapparent until very late in development. Many lines displayed a varietyof other effects such as a reduction in overall size, narrow curledleaves, or various non-specific floral abnormalities, which reducedfertility. These effects on leaf appearance were observed in 18/20primary transformants, and in all the plants from 4/6 of the T2 lines(#2,4,9 and 15) examined. The glossy nature of the leaves from35S::G2583 plants can be a consequence of changes in epicuticular waxcontent or composition. G2583 belongs to a small clade within the largeAP2/EREBP Arabidopsis family that also contains G975 (SEQ ID NO: 19),G1387 (SEQ ID NO: 21), and G977 (SEQ ID NO: 23). Overexpression of G975caused a substantial increase in leaf wax components, as well asmorphological phenotypes resembling those observed in 35S::G2583 plants.G2583 was ubiquitously expressed, at higher levels in root, flower,embryo, and silique tissues. G2583 can be used to modify plantappearance (shiny leaves). In addition, it can be used to manipulate waxcomposition, amount, or distribution, which in turn can modify planttolerance to drought and/or low humidity or resistance to insects.

Closely Related Genes from Other Species

G2583 showed some sequence similarity with known genes from other plantspecies within the conserved AP2/EREBP domain.

G1322: G1322 (SEQ ID NO: 21) was analyzed using transgenic plants inwhich the gene was expressed under the control of the 35S promoter.35S::G1322 transgenic plants were wild-type in phenotype with respect tothe biochemical analyses performed. Overexpression of G1322 producedchanges in overall plant size and leaf development. At all stages,35S::G1322 plants were distinctly smaller than controls and developedcurled dark-green leaves. Following the switch to flowering, the plantsformed relatively thin inflorescence stems and had a rather poor seedyield. In addition, overexpression of G1322 resulted in plants with analtered etiolation response as well as enhanced tolerance to germinationunder chilling conditions. When germinated in the dark, G1322overexpressing transgenic plant lines had open, slightly greencotyledons. Under chilling conditions, all three transgenic linesdisplayed a similar germination response, seedlings were slightly largerand had longer roots. In addition, an increase in the leaf glucosinolateM39480 was observed in all three T2 lines. According to RT-PCR analysis,G1322 was expressed primarily in flower tissue. The utilities of G1322include altering a plant's chilling sensitivity and altering a plant'slight response. The germination of many crops is very sensitive to coldtemperatures. A gene that will enhance germination and seedling vigor inthe cold has tremendous utility in allowing seeds to be planted earlierin the season with a higher survival rate. G1322 can also be useful foraltering leaf glucosinolate composition. Increases or decreases inspecific glucosinolates or total glucosinolate content are desirabledepending upon the particular application. Modification of glucosinolatecomposition or quantity can therefore afford increased protection frompredators. Furthermore, in edible crops, tissue specific promoters canbe used to ensure that these compounds accumulate specifically intissues, such as the epidermis, which are not taken for consumption.

Closely Related Genes from Other Species

G1322 shows some sequence similarity with known genes from other plantspecies within the conserved Myb domain.

G303: The complete sequence of G303 (SEQ ID NO: 23) was determined. G303was detected at very low levels in roots and rosette leaves. G303 wasanalyzed using transgenic plants in which G303 was expressed under thecontrol of the 35S promoter. G303 overexpressing plants showed moretolerance to osmotic stress in a germination assay in three separateexperiments. They showed more seedling vigor than wild-type control whengerminated on plates containing high salt and high sucrose. G303 areuseful for enhancing seed germination under high salt conditions orother conditions of osmotic stress. Evaporation from the soil surfacecauses upward water movement and salt accumulation in the upper soillayer where the seeds are placed. Thus, germination normally takes placeat a salt concentration much higher than the mean salt concentration inthe whole soil profile. Increased salt tolerance during the germinationstage of a crop plant will impact survivability and yield. G303 can alsobe used to engineer plants with enhanced tolerance to drought, saltstress, and freezing.

Closely Related Genes from Other Species

G303 shows some sequence similarity with known genes from other plantspecies within the conserved basic HLH domain.

G1927: G1927 (SEQ ID NO: 239) was analyzed using transgenic plants inwhich the gene was expressed under the control of the 35S promoter.Overexpression of G1927 in Arabidopsis resulted in plants that had analtered response to pathogen. Plants overexpressing G1927 showed fewerdisease symptoms following infection with the fungal pathogenSclerotinia sclerotiorum compared with control plants. The experimentwas repeated on individual lines, and all three lines showed theenhanced pathogen tolerance phenotype. G1927 expression appeared to beubiquitous according to RT-PCR analysis. G1927 can be used to manipulatethe defense response in order to generate pathogen-resistant plants.

Closely Related Genes from Other Species

G1927 showed extensive sequence similarity to a NAC protein from tomato(BG350410).

Example VIII

Identification of Homologous Sequences

Homologous sequences from Arabidopsis and plant species other thanArabidopsis were identified using database sequence search tools, suchas the Basic Local Alignment Search Tool (BLAST) (Altschul et al. (1990)J. Mol. Biol. 215:403–410; and Altschul et al. (1997) Nucl. Acid Res.25: 3389–3402). The tblastx sequence analysis programs were employedusing the BLOSUM-62 scoring matrix (Henikoff, S. and Henikoff, J. G.(1992) Proc. Natl. Acad. Sci. USA 89: 10915–10919).

Identified non-Arabidopsis sequences homologous to the Arabidopsissequences are provided in Table 5. The percent sequence identity amongthese sequences can be as low as 47%, or even lower sequence identity.The entire NCBI GenBank database was filtered for sequences from allplants except Arabidopsis thaliana by selecting all entries in the NCBIGenBank database associated with NCBI taxonomic ID 33090 (Viridiplantae;all plants) and excluding entries associated with taxonomic ID 3701(Arabidopsis thaliana). These sequences are compared to sequencesrepresenting genes of SEQ IDs NOs:2–2N, where N=2–123, using theWashington University TBLASTX algorithm (version 2.0a19MP) at thedefault settings using gapped alignments with the filter “off”. For eachgene of SEQ IDs NOs:2–2N, where N=2–123, individual comparisons wereordered by probability score (P-value), where the score reflects theprobability that a particular alignment occurred by chance. For example,a score of 3.6e-40 is 3.6×10⁻⁴⁰. In addition to P-values, comparisonswere also scored by percentage identity. Percentage identity reflectsthe degree to which two segments of DNA or protein are identical over aparticular length. Examples of sequences so identified are presented inTable 5. Homologous or orthologous sequences are readily identified andavailable in GenBank by Accession number (Table 5; Test sequence ID)Theidentified homologous polynucleotide and polypeptide sequences andhomologues of the Arabidopsis polynucleotides and polypeptides may beorthologs of the Arabidopsis polynucleotides and polypeptides. (TBD: tobe determined.)

Example IX

Introduction of Polynucleotides into Dicotyledonous Plants

SEQ ID NOs: 1–(2N−1), wherein N=2–123, paralogous, orthologous, andhomologous sequences recombined into pMEN20 or pMEN65 expression vectorsare transformed into a plant for the purpose of modifying plant traits.The cloning vector may be introduced into a variety of cereal plants bymeans well-known in the art such as, for example, direct DNA transfer orAgrobacterium tumefaciens-mediated transformation. It is now routine toproduce transgenic plants using most dicot plants (see Weissbach andWeissbach, (1989) supra; Gelvin et al., (1990) supra; Herrera-Estrellaet al. (1983) supra; Bevan (1984) supra; and Klee (1985) supra). Methodsfor analysis of traits are routine in the art and examples are disclosedabove.

Example X

Transformation of Cereal Plants with an Expression Vector

Cereal plants such as corn, wheat, rice, sorghum or barley, may also betransformed with the present polynucleotide sequences in pMEN20 orpMEN65 expression vectors for the purpose of modifying plant traits. Forexample, pMEN020 may be modified to replace the NptII coding region withthe BAR gene of Streptomyces hygroscopicus that confers resistance tophosphinothricin. The KpnI and BglII sites of the Bar gene are removedby site-directed mutagenesis with silent codon changes.

The cloning vector may be introduced into a variety of cereal plants bymeans well-known in the art such as, for example, direct DNA transfer orAgrobacterium tumefaciens-mediated transformation. It is now routine toproduce transgenic plants of most cereal crops (Vasil, I., Plant Molec.Biol. 25: 925–937 (1994)) such as corn, wheat, rice, sorghum (Cassas, A.et al., Proc. Natl. Acad Sci USA 90: 11212–11216 (1993) and barley (Wan,Y. and Lemeaux, P. Plant Physiol. 104:37–48 (1994). DNA transfer methodssuch as the microprojectile can be used for corn (Fromm. et al.Bio/Technology 8: 833–839 (1990); Gordon-Kamm et al. Plant Cell 2:603–618 (1990); Ishida, Y., Nature Biotechnology 14:745–750 (1990)),wheat (Vasil, et al. Bio/Technology 10:667–674 (1992); Vasil et al.,Bio/Technology 11:1553–1558 (1993); Weeks et al., Plant Physiol.102:1077–1084 (1993)), rice (Christou Bio/Technology 9:957–962 (1991);Hiei et al. Plant J. 6:271–282 (1994); Aldemita and Hodges, Planta199:612–617; Hiei et al., Plant Mol Biol. 35:205–18 (1997)). For mostcereal plants, embryogenic cells derived from immature scutellum tissuesare the preferred cellular targets for transformation (Hiei et al.,Plant Mol Biol. 35:205–18 (1997); Vasil, Plant Molec. Biol. 25: 925–937(1994)).

Vectors according to the present invention may be transformed into cornembryogenic cells derived from immature scutellar tissue by usingmicroprojectile bombardment, with the A188XB73 genotype as the preferredgenotype (Fromm, et al., Bio/Technology 8: 833–839 (1990); Gordon-Kammet al., Plant Cell 2: 603–618 (1990)). After microprojectile bombardmentthe tissues are selected on phosphinothricin to identify the transgenicembryogenic cells (Gordon-Kamm et al., Plant Cell 2: 603–618 (1990)).Transgenic plants are regenerated by standard corn regenerationtechniques (Fromm, et al., Bio/Technology 8: 833–839 (1990); Gordon-Kammet al., Plant Cell 2: 603–618 (1990)).

The plasmids prepared as described above can also be used to producetransgenic wheat and rice plants (Christou, Bio/Technology 9:957–962(1991); Hiei et al., Plant J. 6:271–282 (1994); Aldemita and Hodges,Planta 199:612–617 (1996); Hiei et al., Plant Mol Biol. 35:205–18(1997)) that coordinately express genes of interest by followingstandard transformation protocols known to those skilled in the art forrice and wheat Vasil, et al. Bio/Technology 10:667–674 (1992); Vasil etal., Bio/Technology 11:1553–1558 (1993); Weeks et al., Plant Physiol.102:1077–1084 (1993)), where the bar gene is used as the selectablemarker.

All references, publications, patent documents, web pages, and otherdocuments cited or mentioned herein are hereby incorporated by referencein their entirety for all purposes. Although the invention has beendescribed with reference to specific embodiments and examples, it shouldbe understood that one of ordinary skill can make various modificationswithout departing from the spirit of the invention. The scope of theinvention is not limited to the specific embodiments and examplesprovided.

1. A method for increasing the tolerance of a plant to nitrogen limitingconditions as compared to a wild-type plant of the same species, themethod steps comprising: (a) inserting a recombinant polynucleotidehaving a nucleotide sequence encoding a polypeptide comprising aconserved domain with at least about 80% sequence identity to aconserved domain of amino acid coordinates 39–76 of SEQ ID NO: 52 intoan expression vector, wherein the conserved domain is a DNA-bindingdomain; (b) introducing the expression vector into a target plant orplant cell to generate at least one transformed plant, wherein thepolypeptide is overexpressed in the at least one transformed plant andsaid overexpression of the polypeptide results in the at least onetransformed plant having greater tolerance to the nitrogen limitingconditions as compared to the wild-type plant; and (c) from the at leastone transformed plant, identifying a transgenic plant having greatertolerance to the nitrogen limiting conditions than the wild-type plantas a result of the overexpression of the polypeptide in the transgenicplant.
 2. The method of claim 1, wherein the recombinant polynucleotidecomprises a constitutive, inducible, or tissue-specific promoteroperably linked to the nucleotide sequence encoding the polypeptide. 3.The method of claim 1, wherein the polypeptide has a conserved domainwith at least about 85% sequence identity to the conserved domain ofamino acid coordinates 39–76 of SEQ ID NO:
 52. 4. The method of claim 1,wherein the target plant is a dicot.
 5. The method of claim 1, whereinthe target plant is a monocot.
 6. The method of claim 1, wherein thetarget plant is a gymnosperm.
 7. A method for producing a transgenicplant having greater tolerance to nitrogen limiting conditions than awild-type plant of the same species, the method steps comprising: (a)inserting a recombinant polynucleotide having a nucleotide sequenceencoding a polypeptide comprising a conserved domain with at least about80% sequence identity to a conserved domain of amino acid coordinates39–76 of SEQ ID NO: 52 into an expression vector, wherein the conserveddomain is a DNA-binding domain; (b) introducing the expression vectorinto a target plant or plant cell to generate at least one transformedplant, wherein the polypeptide is overexpressed in the at least onetransformed plant and said overexpression of the polypeptide results inthe at least one transformed plant having greater tolerance to thenitrogen limiting conditions as compared to the wild-type plant; and (c)from the at least one transformed plant, identifying a transgenic planthaving greater tolerance to the nitrogen limiting conditions than thewild-type plant as a result of the overexpression of the polypeptide inthe transgenic plant.
 8. The method of claim 7, wherein the recombinantpolynucleotide comprises a constitutive, inducible, or tissue-specificpromoter operably linked to the nucleotide sequence encoding thepolypeptide.
 9. The method of claim 7, wherein the polypeptide has aconserved domain with at least about 85% sequence identity to theconserved domain of amino acid coordinates 39–76 of SEQ ID NO:
 52. 10.The method of claim 7, wherein the transgenic plant has more root growthand is larger than the wild-type plant on MS medium containing as thenitrogen source NH₄NO₃ reduced to 20 mg/l.
 11. The method of claim 7,wherein the expression of the polypeptide results in increased yield inthe transgenic plant relative to the wild-type plant.
 12. The method ofclaim 7, wherein the transgenic plant produces a seed comprising therecombinant polynucleotide of claim 7, and a progeny plant germinatedfrom the seed has greater tolerance to the nitrogen limiting conditionsthan the wild-type plant.